Gapdh

Total protein extracts from HCCC-9810, RBE, QBC-939 and HuCC-T1 cells were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% solution of skim milk powder and TBST, the membranes were incubated overnight with primary antibodies against UBA3, ANXA2, N-Cadherin, Vimentin, MEK, ERK,
^Ser217/221P-MEK
^{Thr158/Tyr187}P-ERK or GAPDH (Abmart, Shanghai, China), followed by incubation with the secondary antibody (ABMT-PT; Abmart) for 1.5 h at room temperature. Finally, protein bands were visualized with P-ECL luminescent solution (Epizyme Biomedical Technology, Shanghai, China) and band density was analyzed using ImageJ. GAPDH was used as the loading control.

Cells and tissue samples were lysed in RIPA buffer with PMSF as protease inhibitor (Beyotime, Shanghai, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on NC or PVDF membrane (Millipore, Bedford, MA). After blocking with 5% dry milk in TBST for 2 h at room temperature, the membranes were incubated with the primary antibodies against SDHB (1:1000, Epitomics), β-tubulin (1:4000, Epitomics), β-actin (1:500, Abmart), caspase 3 (1:1000, Cell Signalling Technology), Bcl-2 (1:4000, Epitomics), MMP-2 (1:500, Abcam), FAK (1:1000, CST), p-FAK (1:1000, CST), AMPKα (1:1000,CST), p-AMPKα (1:1000, CST), GAPDH (1:4000, Abmart), P38 (1:1000, CST), p-P38 (1:1000, CST), ERK (1:1000, CST), p-ERK (1:1000, CST), HIF-1α (1:1000, Epitomics) in dilution buffer overnight at 4°C. Membranes were washed for three times with TBST, then were incubated with IRDye 800CW conjugated goat (polyclonal) anti-Rabbit IgG or anti-Mouse IgG (1:10000) antibodies for 1 h at room temperature. The expression of specific proteins was detected through the use of Odyssey system following the manufacturer's instructions.

Puromycin was purchased from Sigma‐Aldrich. GAPDH, β‐actin, GFP, OFP, SHP2, p‐LCK, LCK, p‐AKT (Thr308), AKT, p‐ZAP70, and ZAP70 antibodies for western blot were purchased from Abmart. FGL1 antibodies were purchased from Santa Cruz Biotechnology Inc. Antibodies, including human PD‐L1 and PD‐1 were purchased from Invitrogen. Human LAG‐3 and Na⁺K⁺ATPase antibodies were purchased from Cell Signaling Technology and Santa Cruz Biotechnology Inc. respectively. Marker antibodies for exosomes, including anti‐CD9, anti‐CD63, and anti‐ALIX, were purchased from Santa Cruz Biotechnology Inc, and anti‐CD81 from System Biosciences. Wheat Germ Agglutinin (WGA) Alexa Fluor 488 and 350 dyes were purchased from Thermo Scientific. Ficoll Paque Plus used for isolating peripheral blood mononuclear cells (PBMC) cells was purchased from GE Healthcare. Staining antibodies, including CD3, CD4, CD8, CD25, and Foxp3 for FACS analysis were purchased from Biolegend Inc.

Tissue and cultured cardiomyocytes were lysed in 1X lysis buffer (Cell Signaling Technology), protein concentration was measured by bicinchoninic acid assay (Pierce), and extracts were subjected to SDS-PAGE using Novex Tris-Glycine Gels (4–20% gradient gel, Life Technologies) or Mini-protean TGX Gels (4–20% gradient gel, Bio-Rad), transferred to nitrocellulose membranes, and probed with various primary antibodies from Cell Signaling Technology: CHIP (#2080S), Hsc70 (#8444S), Myc (#2276S), GAPDH (#2118S), and α-tubulin (#3878S), AbMart Inc.: p-CHIP S20 (Custom antibody, Shanghai, China; Project: 25011–1), Sigma: ubiquitin antibody (#SAB4503053), and Li-Cor: fluorescence-labeled secondary antibodies (#926-32211, 926-32210, 926-68023, or 926-68022). Gels were imaged (Odyssey, Li-Cor) and band intensity quantified (Odyssey Software 3.1).

RPMI-1640 and DMEM medium and foetal bovine serum (FBS) were obtained from GIBCO (Grand Island, NY). Trizol, PrimeScript RT Master Mix and SYBR green PCR master mix were purchased from Takara (Shiga, Japan). Antibodies against LC-3I/II, p62, VPS34, and HDAC6 were purchased from Cell Signaling Technology (Danvers, USA), antibodies against GFP, GRP78 and acetyl-lysine were from Abcam (Cambridge, UK), and anitbodies against β-tubulin, β-actin and GAPDH were from Abmart (Shanghai, China). The anti-CD63 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, USA), and the anti-FITC-, -TRITC-, -Cy5- and -HRP-conjugated secondary antibodies as well as ER-Tracker and Lyso-Tracker were obtained from Invitrogen (Carlsbad, USA). Sodium butyrate, vorinostat (SAHA), tubastatin A and 3-methyladenine were obtained from Cayman Chemical (MI, USA). Anti-FLAG M2 mAb and anti-FLAG M2 agarose were obtained from Sigma (St. Louis, USA).

Cell lysate was prepared using RIPA buffer with protease inhibitors and quantified using the BCA protein assay (BioTek, China). Protein (20 μg) was loaded onto a 10% SDS-PAGE gel that was then transferred onto PVDF membrane and incubated with anti-TGFβR2 (Bioworld Technology, MN.US), anti-TGF-β1 (Cell Signaling Technology), anti-PI3K (Cell Signaling Technology), anti-p-Akt (p-Ser473, Abzoom), anti-c-myc (Santa Cruz), anti-E2F1 (Santa Cruz), anti-CCND1 (Santa Cruz), anti-p21 (Santa Cruz), anti-E-cadherin (Bioworld Technology, MN.US), anti-Vimentin (Bioworld Technology, MN.US), and anti-Snail (Bioworld Technology, MN.US) at 4°C overnight in blocker (3% non-fat dry milk/BSA in TTBS) followed by incubation with HRP-conjugated secondary anti mouse (ZSGB-Bio, China). Protein was normalized with GAPDH (Abmart).