Fast silver stain kit

Lysates from SK-HEP1 cells expressing Flag-METTL3 were prepared using 0.3% NP-40 lysis buffer (0.2 mmol/L EDTA, 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, and 0.3% Nonidet P-40) containing the protease inhibitor cocktail (Roche). Anti-Flag Tag Affinity beads (BioLegend) were incubated with the cell extracts for 12 h at 4°C. After binding was completed, the beads were washed with cold 0.1% NP-40 lysis buffer. Flag peptide (Sigma) was then applied to the beads to elute the Flag protein complex. The eluents were collected and visualized through 10% SDS-PAGE. Silver staining was subsequently performed using a Fast Silver Stain Kit (Beyotime). Distinct protein bands were retrieved and analyzed through LC/MS-MS.

Western blotting (WB) and co-immunoprecipitation (co-IP) were performed as previously described [22 (link)].
The Flag-tagged USP35 expression plasmids or the empty vectors were transfected into the HCT116 cell line. The USP35-associated proteins were co-immunoprecipitated with anti-Flag antibody. The proteins were then separated by SDS-PAGE and stained with Fast Silver Stain Kit (Beyotime Biotechnology). The band of interest was cut for mass spec. analysis according to previously mentioned protocol [22 (link)].

The silver staining procedure was conducted using the Fast Silver Stain Kit (Beyotime) per the instructions. The proteins were identified and quantified using PEAKS software (Bioinformatics Solutions, Inc.). A protein with an area ratio (sense/anti‐sense FC) > 2 and unique peptides >2 was deemed significant.

Co-IP was performed as previously described [27 (link)]. Briefly, the cells were lysed and centrifuged for the supernatant. One tenth of the supernatant was retained for the immunoblot of input, and the rest was incubated with anti-STAT3 (Abcam, ab119352), anti-GNAS (Proteintech, 10,150–2-AP), anti-Flag (Abcam, ab205606), anti-Myc (Abcam, ab32), or rabbit/mouse IgG at 4 °C overnight, followed by further incubation with 10 μl of protein A-agarose beads (Cell Signaling Technology) for another 4 h. The bound proteins were subjected to washing three times for 30 min each and then eluted by boiling for 5 min in the loading buffer. Immunocomplexes were analyzed by SDS-PAGE electrophoresis and Western blotting, and the gel was then stained with the Fast Silver Stain Kit (Beyotime, Shanghai, China). Proteins specially interacting with STAT3 were identified by reverse-phase liquid chromatography coupled with tandem mass spectrometry (ACQUITY UPLC UPLC-QTOF).

RNA pull-down assays were performed essentially as previously described [14 (link)]. Briefly, biotin-labeled TPTEP1 (Sense) or unrelated fragments (Antisense) were obtained by in vitro transcription and biotin RNA labeling mix (Roche, Switzerland). Then the equal amount of biotin-labeled TPTEP1 or unrelated RNA was added as a control to streptavidin dynabeads. QGY-7703 cells lysates were incubated at room temperature for 15 min to immobilize RNA on the streptavidin dynabeads, then supernatant was removed and beads were washed with wash buffer. Samples were then boiled for 10 min at 100 °C in loading buffer. Finally, the samples were subjected into SDS-PAGE and the gel was then stained with the Fast Silver Stain Kit (Beyotime, Shanghai, China). Proteins specially interacting with Lnc TPTEP1 were identified by reverse-phase liquid chromatography coupled with tandem mass spectrometry (ACQUITYTM UPLC-QTOF).

Primary cardiomyocytes were treated with ISO or DMSO (as control) for 24 h. Then, the cells were lysed with Cell lysis buffer for Western and IP (Beyotime, China), and centrifuged to collect the supernatant. One tenth of the supernatant was retained for the input immunoblot, while the rest (300 μg proteins) was incubated with anti-YTHDF2 or rabbit IgG at 4 °C overnight, followed by further incubation with 10 μl protein A/G-agarose beads (Cell Signaling Technology, USA) for another 4 h. The bound proteins were subjected to washing three times for a total of 30 min and then eluted by boiling for 5 min in the loading buffer. Immunocomplexes were subjected into SDS-PAGE electrophoresis and the gel was then stained with the Fast Silver Stain Kit (Beyotime, Shanghai, China). Then, the gel was analyzed independently by reverse-phase liquid chromatography coupled with tandem mass spectrometry using ACQUITYTM UPLC-QTOF analysis platform at Beijing Protein Innovation (Beijing, China). For detecting the interaction between YTHDF2 and MYH7/SRF/BRCA1, the immunoprecipitated proteins were resolved by SDS-PAGE and detected by Western blotting with anti-bodies against MYH7 (22,280–1-AP, ProteinTech, China), SRF (ab252868, Abcam, USA), or BRCA1 (ab238983, Abcam, USA).