P1087

Cells were lysed in RIPA lysis buffer (P10013B, Beyotime) with protease inhibitor (P1008, Beyotime) and phosphatase inhibitor (P1087, Beyotime). Total protein concentration was measured by the Bradford assay and separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, then transferred to polyvinylidene fluoride membrane (Millipore). The membrane was blocked with 5% skimmed milk at room temperature (RT) for 1 h and probed with primary antibodies and subsequently incubated with HRP-conjugated secondary antibodies. The antibodies used in this study are listed as follows. Primary antibodies: anti-Ssrp1 (ab137034, Abcam), anti-GAPDH (HC301, TransGen), anti-β-Tublin (66240-1, Proteintech), anti-phospho-Histone H2A.X (ab11175, Abcam), anti-Spt16 (sc-165987, Santa Cruz). Secondary antibodies: goat anti-mouse IgG-HRP (ab97040, Abcam) and goat anti-rabbit IgG-HRP (ab97051, Abcam).

Cell protein was obtained using RIPA buffer (P0013C; Beyotime) supplemented with protease inhibitor and phosphatase inhibitor (P1082 and P1087; Beyotime). The protein was separated by 8% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred to the enhanced chemiluminescence nitrocellulose membrane. The membranes were first blocked and then incubated with various primary antibodies followed by secondary antibodies (horseradish peroxidase‐conjugated antibody). Primary antibodies were used, including p‐PI3K (17366S; CST, MA, USA), p‐Akt (4060; CST), p‐mTOR (2974T; CST), p‐GSK‐3β (5558; CST), p‐TSC2 (23402S; CST), p‐Raf (9427S; CST), p‐MEK(9154S; CST), p‐ERK (4370S; CST), p‐RSK (11989S; CST), p‐MSK(9595S; CST), CPT1A (ab128568; Abcam, Cambridge, UK), CPT1B (ab134135; Abcam), BIP (3177; CST), CHOP (2895; CST), ACC (12589T; CST), FASN (12589T; CST), PI3K (3358; CST), Akt (4685; CST), mTOR (2983; CST), ERK (4695; CST) and GAPDH (AF1186; Beyotime). HRP‐linked anti‐mouse or rabbit IgG (7076 or 7074; CST) was used as secondary antibodies. The immunoreactivity was shown by Immobilon (P90719; Millipore, MA, USA). The gray value of the western bolt calculated by ImageJ.