Materials and reagents

Materials

Cell culture dish, 100 mm diameter (NEST, catalog number: 704001)
12-well plate (NEST, catalog number: 712011)
1,000 μL blue tip (Biosharp, catalog number: BS-1000-T)
200 μL yellow tip (Biosharp, catalog number: BS-200-T)
10 μL clear tip (Biosharp, catalog number: BS-10-T)
1.5 mL tube (AXYGEN, catalog number: AXYMCT150CS)
0.6 mL tube (AXYGEN, catalog number: AXYMCT060C)
Medical X-ray film (FUJIFILM, catalog number: SUPER RX-N)
Cell scraper (BIOLGIX, catalog number: 70-1180)

Cells

A549 cells (American Type Culture Collection)

Antibodies and plasmids

Anti-Tyk2 antibody (Cell Signaling Technology, catalog number: 14193; 1:1,000)
Anti-AHI1 antibody (Santa Cruz, catalog number: sc-515382; 1:500)
Anti-tubulin antibody (Proteintech, catalog number: 66031-1-Ig; 1:3,000)
Goat anti-rabbit IgG (H+L) HRP (Bioworld, catalog number: BS13278; 1:10,000)
Goat anti-mouse IgG (H+L) HRP (Bioworld, catalog number: BS12478; 1:10,000)
shCtrl plasmid (RNAi-Ready pSIREN-RetroQ-ZsGreen vector)
shAHI1 plasmid
shAHI1(#1)-forward: 5'-CACCGCGGAGACATTATCCGAGTGTTCGAAAACACTCGGATAATGTCTCCG-3'
shAHI1(#1)-reverse: 5'-AAAACGGAGACATTATCCGAGTGTTTTCGAACACTCGGATAATGTCTCCGC-3'
shAHI1(#2)-forward: 5'-CACCGCCATATTGGTCCGACAGTTTCGAAAAACTGTCGGACCAATATGGC-3'
shAHI1(#2)-reverse: 5'-AAAAGCCATATTGGTCCGACAGTTTTTCGAAACTGTCGGACCAATATGGC-3'

Reagents

Dulbecco’s modified Eagle medium (DMEM basic medium) (Gibco, catalog number: C11995500BT)
Fetal bovine serum (FBS) (PAN, catalog number: P30-3302)
Penicillin-streptomycin (Biosharp, catalog number: BL505A)
0.25% trypsin-EDTA (Biosharp, catalog number: BL512A)
Cycloheximide (CHX) (Sigma, catalog number: C7698-1G)
SuperSignal West Dura Extended kit (Thermo Scientiﬁc)
LongTrans (UCallM, catalog number: TF07)
Dimethyl sulfoxide for cell culture (DMSO) (panreac applichem, catalog number: 67-68-5)
Bradford (Sangon Biotech, catalog number: C900164-0200)
Non-fat milk (Biosharp, catalog number: BS102-500g)
BSA (Solarbio, catalog number: A8020)
Serum-free cell freezing medium (NCM Biotech, catalog number: C40100)
KCl (Sangon Biotech, catalog number: 7447-40-7)
NaCl (Sangon Biotech, catalog number: 7647-14-5)
EDTA (Solarbio, catalog number: 9002-07-7)
NP40 (Solarbio, catalog number: 9016-45-9)
Inhibitor cocktail (Sigma, catalog number: 58914000)
NaF (Sangon Biotech, catalog number: 7681-49-4)
PMSF (Solarbio, catalog number: 329-98-6)
Acr-Bis (30%) (Biosharp, catalog number: BL513B)
TEMED (Beyotime, catalog number: SY728)
APS (Sangon Biotech, catalog number: 7727-54-0)
SDS (Solarbio, catalog number: 151-21-3)
Glycine (Solarbio, catalog number: 56-40-6)
Tris base (Solarbio, catalog number: 77-86-1)
Tween 20 (Sangon Biotech, catalog number: 9005-64-5)
β-mercaptoethanol (Sigma, catalog number: 60-24-2)
Isopropyl alcohol (Sangon, catalog number: A507048-0500)
PageRuler prestained protein ladder (Thermo Fisher Scientific, catalog number: 26616)
Na₃VO₄ (Sangon Biotech, catalog number: 13721-39-6)
Na₂HPO₄ (Solarbio, catalog number: 7558-79-4)
KH₂PO₄ (Solarbio, catalog number: 7778-77-0)
Bromophenol blue (Solarbio, catalog number: 34725-61-6)
Glycerol (Solarbio, catalog number: G8190-500 ml)
HCl (Yonghua, catalog number: 210101204)
10× PBS Buffer (see Recipes)
Lysis buffer (1% NP40) (see Recipes)
8% separation gel (see Recipes)
5% stacking gel (see Recipes)
10× running buffer (see Recipes)
10× transfer buffer (see Recipes)
100 mM PMSF (see Recipes)
10% APS (see Recipes)
Stacking mix (see Recipes)
3× loading buffer (see Recipes)
SDS-sample buffer (see Recipes)
1× PBST buffer (see Recipes)
1% NP40 washing buffer (see Recipes)
1 M Tris-HCl (see Recipes)

Equipment

37 °C, 5% CO₂ forced-air incubator (Thermo Scientific, catalog number: 3111)
Electric thermostatic water tank (Shanghai Jing Hong, catalog number: DK-8D)
Dry thermostat (AOSHENG, catalog number: MK-10)
SDS-PAGE gel casting apparatus (Tanon, catalog number: VE-180)
Protein electrophoresis and blotting instruments (Tanon, catalog number: EPS 300)
Centrifuge (Eppendorf, catalog number: 5425R)

Software

Microsoft PowerPoint
ImageJ
GraphPad Prism

Procedure

CHX can be used to determine changes in protein stability/half-live between different conditions, such as knockout vs. wild-type, overexpressed or in a knock-down situation. This protocol uses the example of Tyk2 stability in AHI1 knock-down cells.

Cell culture and transfection
Cells were cultured at 37 °C under 5% CO₂ in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin (complete medium). Cells were cultured with 0.25% trypsin-EDTA enzyme digesting technique and passaged in vitro.
1. Take A549 cells from -80 °C or liquid nitrogen and quickly place them in a 37 °C constant temperature water bath for approximately 2–3 min until the cells are completely thawed.
2. Place the cells into a centrifuge at 157× g for 3 min.
3. Remove the serum-free cell freezing medium, add 1 mL of complete medium to resuscitate cells, and transfer cells to a 100 mm culture dish.
4. Culture A549 cells in a 100 mm culture dish until 90% confluent.
5. Seed the cells at the ratio of 1:24 (5 × 10⁵ cells) into six wells of a 12-well plate.
6. After 18–24 h, observe the cell density (approximately 80%–90%), and then perform the transfection of plasmids (such as shCtrl or shAHI1).
7. Remove the medium and add 1 mL of fresh complete medium before transfection.
8. Label 1.5 mL tubes as required and add 50 μL of DMEM basic medium to the tubes.
9. Prepare transfection solution A: add 1 μg of plasmid into DMEM medium and gently mix in the 1.5 mL tubes.
10. Prepare transfection solution B: add the LongTrans (DMEM:plasmid:longtrans = 50 μL:1 μg:2 μL) into DMEM medium and gently mix in the 1.5 mL tubes.
11. Gently mix transfection solutions A and B and leave at room temperature (RT) for 10–15 min.
12. Gently add the mixture to the corresponding medium and shake gently.
13. Place the plates in the incubator for approximately 36–48 h (for overexpressed plasmid) or 48–72 h (for knockdown plasmid).
14. Treat cells with CHX (50 μg/mL) for the appropriate timepoints (Section B).

CHX treatment
CAUTION: CHX is a highly toxic chemical and very toxic if swallowed; please avoid direct contact and release to the environment. In case of accident or if you feel unwell, seek medical advice immediately.
1. Remove the cell medium and replace with 1 mL of complete medium.
2. Distribute 500 μg of CHX into 0.6 mL tubes.
3. Add 10 μL of DMSO to the tube and mix well (the final concentration of CHX should be 50 mg/mL). CHX is better dissolved fresh to ensure maximal activity.
4. Slowly add 1 μL of CHX into the 1 mL cell culture medium and shake the plate gently to mix well. Similarly, add 1 μL of DMSO into the corresponding well as a control.
5. Harvest cells and extract cellular proteins (Section C).

Extracting cellular proteins
1. Prepare cell lysis buffer: add PMSF (1:100) to 1× NP-40 lysis buffer before use.
2. Place the cells on ice and aspirate the medium.
3. Gently add 1× PBS to wash and discard the PBS thoroughly. Repeat twice.
4. Add lysis buffer to the cells (100 μL per well).
5. Place the cells on ice for 30 min.
6. Scrap down the fully lysed cells with a cell scraper.
7. Transfer cell lysates to a 1.5 mL tube.
8. Place the 1.5 mL tubes into a 4 °C centrifuge at 12,000× g for 15 min.
9. Transfer the supernatant to a new 1.5 mL tube to get the cleared lysate (save at -80 °C); discard the cell debris sediment.
10. Use Bradford to determine protein concentration.
11. Denature 30–50 μg of proteins and add SDS-sample buffer to the samples.
12. Boil the samples in a metal bath at 96 °C for 10 min.
13. Centrifuge at 12,000× g for 1 min.
14. Use the samples for western blot or save them at -20 °C for future use.

Protein degradation analysis by western blot
1. Prepare the 8% SDS-PAGE gels (the gel concentration can be adjusted to 8%–12% depending on the molecular weight of the detected protein) with 8% separation gel and 5% stacking gel.
2. Dilute the 10× running buffer to 1× with double-distilled water (ddH₂O).
3. Add 1× running buffer to the vertical electrophoresis chamber.
4. Gently pull out the comb of SDS-PAGE gel under running water and assemble it into vertical electrophoresis chamber.
5. Fill the chamber with running buffer.
6. Load the denatured protein samples into the lane of stacking gel and place 2 μL of PageRuler prestained protein ladder on both sides to indicate the molecular weight of the proteins.
7. Run for approximately 30 min using constant 80 V when the samples are in the stacking gel, and for approximately 90 min in the resolving gel at 110 V for a better separation of proteins.
8. Soak the PVDF membrane in absolute methanol for approximately 1 min to activate.
9. Prepare 1× transfer buffer (10× transfer buffer:absolute methanol:double-distilled water = 1:1:8).
10. Put the gel onto the activated PVDF membrane in 1× transfer buffer in the sequence of sponge, filter paper, gel, filter paper, and sponge.
11. Transfer the proteins to the gel using constant 360 mA for 90–120 min and immerse the electrophoresis tank in ice water.
12. Block the PVDF membrane with 5% non-fat milk with gentle shaking for 30 min at RT.
13. Dilute primary antibodies in 1% BSA at the indicated dilution.
14. Incubate the membrane with diluted primary antibodies with gentle shaking at RT for 2 h or 4 °C overnight.
15. Wash the membrane in 1× PBST buffer at RT for 10 min. Repeat three times.
16. Dilute the anti-mouse or anti-rabbit secondary antibodies in 5% non-fat milk at 1:10,000.
17. Incubate the membrane with the secondary antibodies with gentle shaking at RT for 1 h.
18. Wash the membrane in 1× PBST buffer at RT for 10 min. Repeat three times.
19. Visualize immunoreactive bands with a SuperSignal West Dura Extended kit using X-ray film in the dark room.
20. Edit gel images to the right size using Microsoft PowerPoint.
21. Use ImageJ to count pixels of the bands and calculate protein half-life.
22. Use GraphPad Prism to make the line chart.

Data analysis

Figure 1 displays a representative result showing the protein stability of Tyk2 in A549 cells transfected with shCtrl or shAHI1.

Figure 1. AHI1 knockdown accelerated Tyk2 protein degradation. (A) A549 cells were transfected with either control shRNAs (shCtrl) or shRNAs against AHI1 (shAHI1) plasmids. Seventy-two hours after transfection, cells were treated with either DMSO (for 0 h timepoint) or CHX (50 μg/mL, for 6 h and 12 h timepoints). Whole-cell extracts were analyzed by western blot using the antibodies against Tyk2, AHI1, or Tubulin. Tubulin serves as a loading control. (B) The striped images of Tyk2 and Tubulin were imported into Image J. The pixel count of all the strips was measured, and then the percentage of Tyk2 that remained was calculated in GraphPad Prism software. From Zhang et al. (2022).

Notes

The degradation rates of proteins can vary considerably from one another. Thus, it is suggested to determine the time course of protein degradation using the CHX chase assay in advance. Some endogenous proteins degrade very rapidly, and therefore the treatment time of CHX can be adjusted to 30–60 min. Sometimes it is necessary to set as few as five timepoints to detect the half-life of the protein. You can also find other experimental data about CHX chase assay in other papers of our research group (Feng et al., 2018; Yuan et al., 2020).

Recipes

10× PBS buffer
Reagent Amount (g)
NaCl 80.00
KCl 2.00
Na₂HPO₄ 14.20
KH₂PO₄ 2.70
Dilute the 10× PBS buffer to 1× PBS with ddH₂O.

Lysis buffer (1% NP40)
Reagent Volume (mL)
1% NP40 washing buffer (Recipe 13) 9.85
EDTA (0.5 M) 0.05
Na₃VO₄ (0.2 M) 0.05
NaF (0.5 M) 0.02
Inhibitor cocktail 0.02

8% separation gel
Reagent Volume (mL)
ddH₂O 3.80
Tris-HCl (pH 8.0) (Recipe 14) 2.00
Acr-Bis (30%) 2.13
10% APS (Recipe 8) 0.08
TEMED 8.00 × 10^-3

5% stacking bel
Reagent Volume (mL)
Stacking mix (see Recipe 9) 3.00
10% APS (Recipe 8) 0.02
TEMED 5.00 × 10^-3

10× running buffer
Reagent Amount (g)
Tris base 30.00
Glycine 144.00
SDS 40.00
Dilute the 10× running buffer to 1× running buffer with ddH₂O

10× transfer buffer
Reagent Amount (g)
Tris base 24.00
Glycine 142.00
Dilute the 10× transfer buffer to 1× transfer buffer with ddH₂O

100 mM PMSF
Reagent Amount
PMSF 0.696 g
Isopropyl alcohol 40.00 mL

10% APS
Reagent Amount
APS 1.00 g
ddH₂O 10.00 mL

Stacking mix
Reagent Volume (mL)
Acr-Bis (30%) 16.70
1 M Tris-HCl (pH 6.8) (Recipe 14) 12.50
20% SDS 0.50
ddH₂O 70.30

3× loading buffer
Reagent Amount
1 M Tris-HCl (pH 6.8) (Recipe 14) 1.88 mL
20% SDS 3.00 mL
Glycerol 3.00 mL
Bromophenol blue 6.00 × 10^-3g
ddH₂O 2.20 mL

SDS-sample buffer
Reagent Ratio (%)
3× loading buffer (Recipe 10) 85
β-mercaptoethanol 15

1× PBST buffer
Reagent Volume (mL)
1× PBS 500
Tween 20 5

1% NP40 washing buffer
Reagent Volume (mL)
5 M NaCl 1.50
1 M Tris-HCl (pH 7.4) 2.50
NP40 0.50
ddH₂O 45.50

1 M Tris-HCl
Reagent Amount
Tris 121.10 g
HCl to pH = 6.8, 7.4, or 8.0
ddH₂O to 1 L

Acknowledgments

This work was supported by grants from the Project of Natural Science Research in Colleges and Universities of Jiangsu Province (20KJB310008), the Foundation for Young Scholars of Jiangsu Province (BK20200864), and the National Key R&D Program of China (2018YFC1705500): 2018YFC1705505. This protocol was adapted from previous work published in Cell Research (Zhang et al., 2022).

Competing interests

The authors declare no conﬂict of interest.

Ethics

The study did not involve human subjects or animal work.

References

Feng, Q., Miao, Y., Ge, J., Yuan, Y., Zuo, Y., Qian, L., Liu, J., Cheng, Q., Guo, T., Zhang, L., Yu, Z. and Zheng, H. (2018). ATXN3 Positively Regulates Type I IFN Antiviral Response by Deubiquitinating and Stabilizing HDAC3. J Immunol 201(2): 675-687.
Fritzsche, S. and Springer, S. (2014). Pulse-chase analysis for studying protein synthesis and maturation. Curr Protoc Protein Sci 78: 30 33 31-30 33 23.
Glickman, M. H. and Ciechanover, A. (2002). The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82(2): 373-428.
Goldberg, A. L. (2003). Protein degradation and protection against misfolded or damaged proteins. Nature 426(6968): 895-899.
Hanzl, A. and Winter, G. E. (2020). Targeted protein degradation: current and future challenges. Curr Opin Chem Biol 56: 35-41.
Huang, T., Song, X., Yang, Y., Wan, X., Alvarez, A. A., Sastry, N., Feng, H., Hu, B. and Cheng, S. Y. (2018). Autophagy and Hallmarks of Cancer. Crit Rev Oncog 23(5-6): 247-267.
Lu, B., Al-Ramahi, I., Valencia, A., Wang, Q., Berenshteyn, F., Yang, H., Gallego-Flores, T., Ichcho, S., Lacoste, A., Hild, M., et al. (2013). Identification of NUB1 as a suppressor of mutant Huntington toxicity via enhanced protein clearance. Nat Neurosci 16(5): 562-570.
Paiva, S. L. and Crews, C. M. (2019). Targeted protein degradation: elements of PROTAC design. Curr Opin Chem Biol 50: 111-119.
Park, H., Kang, J. H. and Lee, S. (2020). Autophagy in Neurodegenerative Diseases: A Hunter for Aggregates. Int J Mol Sci 21(9).
Schneider-Poetsch, T., Ju, J., Eyler, D. E., Dang, Y., Bhat, S., Merrick, W. C., Green, R., Shen, B. and Liu, J. O. (2010). Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nat Cem Biol 6(3): 209-217.
Spradlin, J. N., Zhang, E. and Nomura, D. K. (2021). Reimagining Druggability Using Chemoproteomic Platforms. Acc Chem Res 54(7): 1801-1813.
Sun, T., Liu, Z. and Yang, Q. (2020). The role of ubiquitination and deubiquitination in cancer metabolism. Mol Cancer 19(1): 146.
Yuan, Y., Miao, Y., Qian, L., Zhang, Y., Liu, C., Liu, J., Zuo, Y., Feng, Q., Guo, T., Zhang, L., et al. (2020). Targeting UBE4A Revives Viperin Protein in Epithelium to Enhance Host Antiviral Defense. Mol Cell 77(4): 734-747 e737.
Zhang, H. G., Wang, B., Yang, Y., Liu, X., Wang, J., Xin, N., Li, S., Miao, Y., Wu, Q., Guo, T., et al. (2022). Depression compromises antiviral innate immunity via the AVP-AHI1-Tyk2 axis. Cell Res 32(10): 897-913.