Co ip lysis buffer

For co‐IP assays, protein was immunoprecipitated as previously described (Chen et al., 2017). Briefly, total protein was harvested using co‐IP lysis buffer (Beyotime Biotechnology) supplemented with protease inhibitor (P‐8340; Sigma, St. Louis, MO, USA). The mixture was centrifuged, and the supernatant was incubated with primary antibody or negative control antibody for 4 h on ice. Twenty microliters of protein A/G plus beads (sc‐2003; Santa Cruz) was added and incubated overnight at 4 °C. Precipitates were washed three times with lysis buffer, and western blot was performed to detect the presence of the indicated protein.

MIA‐PaCa‐2 and PANC‐1 cells were lysed in ice‐cold co‐immunoprecipitation (Co‐IP) lysis buffer (Beyotime, Wuhan, China) and were then incubated on ice for 10 minutes. The insoluble material was pelleted at 13 000 × g for 10 minutes at 4°C. The supernatant was pre‐cleaned by protein A/G PLUS‐Agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and the aliquots were immunoprecipitated with an antibody against KLF5 (21017‐1‐AP; Thermo Fisher Scientific), followed by incubation with protein A/G PLUS‐Agarose beads overnight at 4°C. The immunoprecipitated complexes were washed, and the precipitated proteins were then analysed by Western blot analysis as described above. The input was used as a positive control.

NG and HG cells were transfected with shARAP1 or empty vector. After transfection, the cells were treated with the proteasome inhibitor MG132 at 50 μg/mL (MedChemExpress LLC) for 12 hours. The cells were then lysed in co‐IP lysis buffer (Beyotime) and immunoprecipitated with anti‐EGFR antibody overnight at 4°C. Anti‐ubiquitin antibody (#3933, Cell Signaling Technology) was used to detect ubiquitinated EGFR in the precipitates. The precipitates and lysates were analysed by Western blotting to detect the protein expression of ARAP1, EGFR and β‐actin.

We extracted total proteins using co-immunoprecipitation (co-IP) lysis buffer (Beyotime, Beijing, China), cell lysis were precleared with rabbit IgG for 2 h and then incubated with anti-TRIM11 (Proteintech, 10851-1-AP) or anti-YAP (Proteintech, 13584-1-AP) antibody overnight as previously described 32 . And we used rabbit IgG (Santa Cruz) as the negative control. The immunoprecipitated proteins were collected for Western blot analysis and analyzed using Anti-YAP or Anti-TRIM11 antibody.

For western blotting, brain tissues or cells were lysed in RIPA lysis buffer (Solarbio) containing 1% protease inhibitor cocktail and 1% PMSF. Protein concentration was determined by Pierce BCA Protein Assay Reagent. Next, Protein samples were separated by 12% or 10% SDS–PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% milk or BSA in PBST (PBS containing 0.02% Tween‐20) for 1 h at room temperature, and incubated with primary antibodies at 4 °C overnight. The bands were visualized and analyzed by the Image Studio Ver 5.2 software after secondary antibodies for 1.5 h at room temperature.
For co‐immunoprecipitation, protein samples were lysed in co‐IP lysis buffer (Beyotime Biotechnology) containing 1% protease inhibitor cocktail and 1% PMSF. The supernatant was collected by centrifugation at 4 °C, and incubated with anti‐HA‐tag (MBL) or anti‐Flag‐tag magnetic beads (MBL) overnight at 4 °C. After washing three times with cold wash buffer, the bound proteins were analyzed by western blot.

The total protein of HEK 293T cells with different transfections was extracted in Co-IP lysis buffer (Beyotime, Beijing, China). For immunoprecipitation, the cell lysate was incubated with anti-flag (#ab1162, Abcam) or anti-HA (#ab18181, Abcam) or anti-Myc (#ab32) antibody and Dynabeads Protein G at 4 °C overnight. Then, the beads were washed three times with lysis buffer, and the immunoprecipitates were harvested for further western blot analysis.

Co-IP lysis buffer (Beyotime, China) was applied to lyse the cells. Cell lysis was precleared with rabbit IgG for 2 h, and the specified antibody was then immunoprecipitated at 4 °C overnight. The lysates were then mixed with Protein A/G PLUS-Agarose beads (Santa Cruz, USA) and incubated at 4 °C for 2 h. The immunocomplexes were separated by SDS-PAGE after washing three times with lysis buffer and followed by a standard immunoblotting process.