Anti flag affinity gel

HEK293T cells were transfected with 3 × Flag-ELF5 and p300 for 48 h, and then were lysed with TNE buffer. Flag-ELF5 protein was purified by immunoprecipitation with anti-Flag Affinity Gel (Bimake, Houston, USA) and subjected to SDS-PAGE. After SDS-PAGE and Coomassie blue staining, the appropriate bands containing Flag-ELF5 proteins were excised from the gel and collected. Samples were washed three times with 50 mM NH₄HCO₃ in 30% acetonitrile for 20 minutes. Then the samples were incubated with 300 μL acetonitrile for 10 min. After acetonitrile removed, the gels were reduced by incubation with 20 mM dithiothreitol at 56 °C for 30 min. Then, the gels were alkylated by 100 mM iodoacetamide in the dark for 20 min. The dried gels were digested with trypsin overnight and then the protein digest was extracted with 85% acetonitrile and 0.1% trifluoroacetic acid for twice. The remained solution was re-dissolved in 30 μL of 0.1% formic acid and analyzed by LC-MS system (EASY-nLC 1200, Thermo Scientific) coupled to an ion trap spectrometer (LTQ Velos Pro, Thermo Scientific). The resulting MS/MS data were used to search the Swiss-Prot database using Proteome Discoverer (Thermo Fisher Scientific) with MASCOT search engine software (Matrix Science).

Co-IP and LC‒MS/MS experiments were used to determine potential ASS1 interacting proteins. Briefly, 293 T cells were infected with p3XFlag-ASS1-cmv-10 and the empty vector for 48 h. ASS1-3xFlag and 3xFlag were pulled down using an anti-Flag affinity gel (catalog B23101, Bimake), followed by SDS‒PAGE electrophoresis after washing. Silver staining was performed and specific adhesive strips were cut for LC‒MS/MS analysis at the BGI.

Briefly, cells were lysed in NETN lysis buffer. Antibodies against HRD1 and CPT2 were used for immunoprecipitation. For the Flag bead pulldown experiment, HEK 293T cells were transfected with Flag‐tagged protein and lysed in NETN buffer for 20 min at 4 °C. Crude lysates were subjected to centrifugation at 14 500 g for 15 min at 4 °C. Supernatants were incubated with Anti‐Flag Affinity Gel (Bimake, B23102, Houston, TX, USA) for 4 h. The agaroses were washed three times with NETN buffer. Proteins were eluted by boiling in 1× SDS running buffer and subjected to SDS/PAGE for immunoblotting.

Cells or tumor tissue were lysed with RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (Beyotime, Shanghai, China) and phosphatase inhibitors (Bimake, USA). Lysates were denatured by heating for 5 min at 99 °C and loaded on 4-10% SDS-polyacrylamide gel electrophoresis. Then the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked and probed with primary antibodies and secondary HPR-conjugated antibodies. At last the membranes were detected by chemiluminescence (Tanon, Shanghai, China). For co-immunoprecipitation experiments, 293T cells were plated and grew to about 80% confluency, then co-transfected with plasmids of HA-STAT3 and Flag-STAT3 using Lipofectamine 2000. After 24 h, cells were treated with different concentrations of W2014-S for following 24 h, and stimulated with IL-6 (100 ng/mL) for 1 h. Cells were lysed with IP RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (Beyotime, Shanghai, China) and phosphatase inhibitors (Bimake, America), and lysates were cultured with Anti-flag Affinity Gel (B23102, Bimake, USA) overnight at 4 ℃. The gel was washed by PBST for three times and added 1×loading buffer and denatured by heating for 5 min at 99 ℃. Then the proteins were resolved on SDS-PAGE, transferred to PVDF membranes and analysed with immunoblotting.