Materials and Reagents

60 mm dish (Thermo Fisher Scientific, Thermo Scientific Nunc, catalog number: 150288)
1.5 ml Hyper Microtube “High-Seal” (WATSON Bio Lab, catalog number: 131-815C)
Pierce NeutrAvidin coated 96-well white plate (Thermo Fisher Scientific, catalog number: 15116)
BHK-21 cells (ATCC^® CCL-10^TM)
BHK cells expressing HBH (His-Biotin-His) and 3HA tagged ∆F508 CFTR (Okiyoneda et al., 2018)
Antibodies
1^st antibodies
1. Anti-Lys48 specific ubiquitin antibody (Apu2) (Merck KGaA, catalog number: 05-1307)
2. Anti-Lys63 specific ubiquitin antibody (Apu3) (Merck KGaA, catalog number: 05-1308)
3. Anti-HA tag antibody (16B12) (BioLegend, catalog number: 901515)
2^nd antibodies
1. HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch, catalog number: 115-035-166)
2. HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch, catalog number: 711-035-152)
Pepstatin A (Peptid institute, catalog number: 4397)
Leupeptin Hemisulfate monohydrate (FUJIFILM Wako Pure Chemical Corporation, catalog number: 122-03751)
PMSF (FUJIFILM Wako Pure Chemical Corporation, catalog number: 164-12181)
MG132 (Cayman Chemical, catalog number: 10012628)
N-Ethylmaleimide (Wako, catalog number: 054-02063)
NP-40 substitute (FUJIFILM Wako Pure Chemical Corporation, catalog number: 145-09701)
Urea (FUJIFILM Wako Pure Chemical Corporation, catalog number: 217-00615)
BSA (FUJIFILM Wako Pure Chemical Corporation, catalog number: 015-27053)
SuperSignal^TM West Pico Chemiluminescent Substrate (Thermo Fisher, catalog number: 34080)
Tris-HCl (FUJIFILM Wako Pure Chemical Corporation, catalog number: 204-07885)
NaCl (FUJIFILM Wako Pure Chemical Corporation, catalog number: 195-01663)
KCl (FUJIFILM Wako Pure Chemical Corporation, catalog number: 163-03545)
Na₂HPO₄ (FUJIFILM Wako Pure Chemical Corporation, catalog number: 197-02865)
KH₂PO₄ (FUJIFILM Wako Pure Chemical Corporation, catalog number: 169-04245)
SDS (FUJIFILM Wako Pure Chemical Corporation, catalog number: 191-07145)
Triton X-100 substitute (FUJIFILM Wako Pure Chemical Corporation, catalog number: 168-11805)
Sodium Deoxycholate (FUJIFILM Wako Pure Chemical Corporation, catalog number: 194-08311)
PBS (see Recipes)
RIPA buffer (see Recipes)
Lysis buffer (see Recipes)
Wash buffer (see Recipes)
Blocking buffer (see Recipes)
Denaturing buffer (see Recipes)
Urea wash buffer (see Recipes)

Equipment

Varioskan^® Flash (Thermo Fisher Scientific, Thermo Scientific, catalog number: 5250040)
Wellwash^TM Versa Microplate Washer (Thermo Fisher Scientific, Thermo Scientific, catalog number: 5165010)
8 channel electronic pipette (Sartorius, catalog number: 730390) and 50-1,200 μl tips (Sartorius, catalog number: 791204)
Forma^TM 310 Direct Heat CO₂ incubators (Thermo Fisher Scientific, Thermo Scientific, catalog number: 310, set at 37 °C with 5% CO₂)

Software

SkanIt^TM (Thermo Fisher scientific)

Procedure

The scheme of the procedure is provided in Figure 1.

Figure 1. Scheme of the procedure of ELISA based protein ubiquitylation measurement. 1. Immobilize HBH tagged CFTR on NeutrAvidin coated 96-well plate. 2. The CFTR binding proteins (other proteins) are removed by adding the Denaturing buffer and washing with the Urea wash buffer. 3. Using an anti-Ub antibody (e.g., anti-Lys48 specific ubiquitin antibody), the ubiquitylation is detected. Refer to the procedure for a detailed description.

Lysate preparation from BHK cells expressing Biotin-tagged CFTR
1. Prepare confluent BHK cells (negative control) and BHK cells expressing Biotin-tagged target protein (e.g., HBH-∆F508-CFTR-3HA) on a 60 mm dish.
2. Incubate with inhibitors (e.g., 10 μM MG-132) for 3 h at 37 °C to accumulate the ubiquitylation.
3. Lyse cells with 1 ml of lysis buffer (Recipe 3) on ice.
4. Centrifuge (14,000 x g) for 15 min at 4 °C.
5. Transfer 1 ml of supernatant to a fresh tube and place on ice for immediate assay or store at -80 °C for future usage.
Biotin-tagged CFTR immobilization on NeutrAvidin coated 96-well plate
1. Wash the plate with the Wash buffer (400 μl/well) (Recipe 4).
2. Block the plate with the Blocking buffer (100 μl/well) (Recipe 5) for 15-30 min on ice.
3. Add the cell lysate to the plate according to Figure 2.
  Note: Add 50-150 μl/well of cell lysate depending on the desired signal for detection. It is essential to optimize the amount of cell lysate to obtain enough, but not saturated signals by the protein-of-interest. Prepare triplicate or quadruplicate samples for both CFTR and Ub detection. Negative control using cell lysate from BHK cells not expressing HBH-CFTR-3HA is necessary to measure the background signal.
  
  Figure 2. Assay template for ELISA based protein ubiquitylation measurement with negative control and two samples of HBH-∆F508 CFTR-3HA in BHK cells. Cell lysate from BHK cells not expressing HBH-ΔF508 CFTR-3HA is used as a negative control to measure the background signal. BHK cells stably expressing HBH-ΔF508 CFTR-3HA were treated with 10 μM MG-132 for 3 h at 37 °C before the cell lysis.
4. Incubate for 2 h at 4 °C.
5. Wash the plate with the Wash buffer (400 μl/well) once.
  Note: If the target protein tends to aggregate, add detergent in all the steps.
6. Add Denaturing buffer (100 μl/well) (Recipe 6) and incubate for 5 min at RT (Room Temperature).
7. Wash the plate with the Urea wash buffer (400 μl/well) (Recipe 7) for 5 times.
Detection of the CFTR ubiquitylation by ELISA
1. Block the CFTR-immobilized plate with the Blocking buffer (100 μl/well) for 20 min at RT.
2. Add the primary antibody (50 μl/well) diluted with Blocking buffer according to Figure 2, and incubate for 1 h at RT.
  Note: For the primary antibody, the following conditions may work: Anti-Lys48 specific ubiquitin antibody (Apu2) (1:500); Anti-Lys63 specific ubiquitin antibody (Apu3) (1:500).
3. Wash the plate with the Wash buffer (400 μl/well) 4 times.
4. Add 50 μl/well of the secondary antibody diluted with the Blocking buffer according to Figure 2, and incubate for 45-60 min at RT.
  Note: For the secondary antibody, the following conditions may work: HRP-conjugated anti-mouse IgG (1:1000); HRP-conjugated anti-rabbit IgG (1:1000).
5. Wash the plate with the Wash buffer (400 μl/well) 6 times.
6. Add SuperSignal^TM West Pico Chemiluminescent Substrate (100 μl/well) and incubate for 5 min at RT.
7. Read the luminescence signal by SkanIt^TM software in Varioskan^® Flash plate reader (Figure 3).
8. The CFTR ubiquitylation level quantified by the anti-Ub antibody is normalized for the CFTR level quantified by the anti-HA antibody (Figure 4).
  Note: For normalization by the target protein, measure the amount of immobilized protein. If the target protein’s signal is too strong, reduce the amount of cell lysate to prevent the saturation of the signal.
  
  Figure 3. Protocol in SkanIt^TM software. The luminescence signal is measured by the Varioskan^® Flash plate reader with the SkanIt^TM software. The protocol consists of 3 steps (boxed in red). 1^st step is shaking the plate for 5 s. 2^nd step is incubation (Pause) for 3 min. 3^rd step is the luminescence measurement in a second.
  
  Figure 4. A typical result of the HBH-∆F508 CFTR-3HA ubiquitylation in BHK cells by ELISA according to Figure 2. A. The amount of HBH-ΔF508 CFTR-3HA immobilized on the NeutrAvidin plate (8 μg/well cell lysate) was measured using anti-HA antibody. B-C. The amount of K48-linked poly-ubiquitin chains (B) (80 μg/well cell lysate) and K63-linked poly-ubiquitin chains (C) (80 μg/well cell lysate) on the immobilized CFTR was measured using anti-K48-Ub and anti-K63-Ub antibodies, respectively. D-E. The amount of K48-linked poly-ubiquitylation (D) and K63-linked poly-ubiquitylation (E) of HBH-ΔF508 CFTR-3HA was quantitated after the normalization for the CFTR level quantified by the anti-HA antibody.

Data analysis

Calculate the specific signal of the ubiquitin chains (e.g., αK48-Ub) and CFTR (αHA) by subtracting the mean value of the background signal from the negative control.
Calculate the relative level of the specific signal (e.g., αK48-Ub, αHA) of drug-treated cells (e.g., MG-132) compared to the untreated control, as shown in Figures 4A-4C.
Calculate the relative level of ubiquitination by normalizing for the CFTR level (e.g., αK48-Ub/αHA) as shown in Figures 4D and 4E.
For quantification, triplicate or quadruplicate experiments are repeated at least two times, while the data is expressed at means ± SEM. Statistical significance is assessed by two-tailed paired Student’s t-test using Excel software (Microsoft).

Recipes

PBS
137 mM NaCl
2.68 mM KCl
8.1 mM Na₂HPO₄
1.47 mM KH₂PO₄
RIPA buffer
20 mM Tris-HCl (pH 8.0)
150 mM NaCl
0.1% SDS
1% TritonX-100
0.5% Sodium Deoxycholate
Lysis buffer
RIPA buffer
5 μg/ml Leupeptin
5 μg/ml Pepstatin A
100 μM PMSF
20 μM MG132
5 mM N-Ethylmaleimid
Wash buffer
PBS
0.1% NP-40
Blocking buffer
PBS
0.1% NP-40
0.5% BSA
Denaturing buffer (freshly prepared)
PBS
8 M Urea
0.1% NP-40
Urea wash buffer (freshly prepared)
RIPA buffer
2 M Urea

Acknowledgments

This protocol was adapted from Okiyoneda et al. (2018). We thank S. Hansen for carefully reading the manuscript. This work was supported by JSPS KAKENHI grant numbers JP25893275, 15H05643, 15H01192 to T.O.

Competing interests

The authors declare no competing financial interests.

References

Conte, A. and Sigismund, S. (2017). Methods to Investigate EGFR Ubiquitination. Methods Mol Biol 1652: 81-100.
Okiyoneda, T., Veit, G., Sakai, R., Aki, M., Fujihara, T., Higashi, M., Susuki-Miyata, S., Miyata, M., Fukuda, N., Yoshida, A., Xu, H., Apaja, P. M. and Lukacs, G. L. (2018). Chaperone-independent peripheral quality control of CFTR by RFFL E3 ligase. Dev Cell 44(6): 694-708 e697.
Tagwerker, C., Flick, K., Cui, M., Guerrero, C., Dou, Y., Auer, B., Baldi, P., Huang, L. and Kaiser, P. (2006). A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivocross-linking. Mol Cell Proteomics 5(4): 737-748.