Anti glyceraldehyde 3 phosphate dehydrogenase gapdh

The c-Maf and Otub1 plasmids were subcloned into a pcDNA3.1 vector carrying an HA, or Myc tag. The luciferase reporter driven by c-Maf recognition element (MARE) (5′-TGCGAGTGAGGCA-3′) (pGL4-MARE.Luci) was synthesized by GeneWiz, Inc. (Suzhou, Jiangsu, China) [7 (link)]. The Otub1-siRNA and control-siRNA were purchased from Ribobio Inc. (Guangzhou, China) [11 ].
The antibodies used for Western blot were as follows: anti-Otub1 and anti-Ub were from Santa Cruz Biotechnology Co. Ltd (Santa Cruz, CA); anti-glyceraldehyde-3-phosphatedehydrogenase (GAPDH), anti-integrin beta 7 (ITGB7) and anti-c-Maf were from Proteintech Group, Inc. (Wuhan, China); anti-HA and anti-Myc were obtained from MBL Biotech Co., (Beijing, China); anti-poly(ADP-ribose) polymerase (PARP), anti-Caspase 3 and anti-cyclin D2 (CCND2) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies of Otud4, Otud5, and Otud7b were provided by Beyotime Institute of Biotechnology (Haimen, China).

Cell lysates and exosomal lysates were subjected to SDS‐PAGE. Western blot analyses were performed by using anti‐collagen I (1:1 000; 14695‐1‐AP; Proteintech), anti‐α‐smooth muscle actin (SMA) (1:1 000; 14395‐1‐AP; Proteintech), anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (1:1 000; 14497‐1‐AP; Proteintech), anti‐immunoglobulin heavy chain binding protein (BIP) (1:1 000; 11587‐1‐AP; Proteintech), anti‐spliced X‐box binding protein 1 (XBP1s) (1:1 000; #12782; Cell Signaling Technology), and anti‐phospho eukaryotic initiation factor 2 (eIF2) α (P‐eIF2α) (1:1 000; #3398; Cell Signaling Technology) antibodies and corresponding HRP‐conjugated secondary antibodies. A chemiluminescent system (Amersham ECL Plus) was used for detection.

Myricetin, gallic acid, ADP, thrombin, phorbol-12-myristate-13-acetate (PMA), thrombin Receptor Activator Peptide 6 (TRAP-6), human fibrinogen, and 1,4-Dithiothreitol (DTT) and 3,3′-Dihexyloxacarbocyanine iodide (DIOC₆) were purchased from Sigma-aldrich (Dorset, UK). PAPA-NONOate was purchased from Tocris (Abingdon, UK). PE/Cy5 anti human CD62P and PAC-1 FITC antibodies were purchased from BD Biosciences (Wokingham, UK). FITC-conjugated fibrinogen was purchased from Agilent (Stockport, UK). Collagen was purchased from Nycomed (Munich, Germany) whereas Collagen-Related Peptide (CRP) was obtained from Prof Richard Farndale (University of Cambridge, Cambridge, UK). Anti-phospho-vasodilator-stimulated phospho-protein (VASP) (Ser239) was purchased from Cell Signalling (Hitchin, UK), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Proteintech (Manchester, UK), and Alexa-488 conjugated phalloidin secondary antibody was bought from Life Technologies (Paisley, UK)

After cells were transfected with the pSin-EF2-puro-ZNF671-HA or pSin-EF2-puro-vector plasmids (Land Hua Gene Biosciences, Guangzhou, China) for 48 h, Radio-Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime, Shanghai, China) was used to isolate proteins. Proteins were separated by Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime), transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and incubated with primary anti-ZNF671 (1:500; Proteintech, Chicago, IL, USA) and anti-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000, Proteintech, Chicago, IL, USA).

NSCLC cells (2 × 10⁵ cells/well) were seeded in two 6-well plates with corresponding medium, and incubated in normoxic and hypoxic incubators for 48 hours, respectively. Then, the medium was discarded, and the cells were lysed to extract proteins. A bicinchoninic acid (BCA) protein assay kit (Applygen, Beijing, China) was used to quantify the proteins from the NSCLC cells. Subsequently, protein (20 μg/sample) was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and incubated in 5% skim milk for 2 h. Then, the membrane was incubated with primary antibodies at 4 °C overnight: anti-eFI5A2 (1:1000, Abcam, Cambridge, UK, ab126733), anti-HIF-1α (1:1000, CST, Danvers, MA, USA), anti-p62 (1:1000, CSTA), anti-light chain 3 (LC3, 1:1000, CST), anti-Beclin-1 (1:1000, CST), anti-autophagy related 3 (ATG3, 1:1000, CST), anti-Tubulin (1:1000, CST), anti-β-actin (1:1000, CST), and anti-glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, 1:1000, ProteinTech, Chicago, IL, USA). After washing three times, the membrane was incubated with the corresponding secondary antibodies (1:2000, CST) at room temperature for 2 h. GADPH, Tubulin, and β-actin was considered normalization controls. Finally, the immunoreactive bands were detected using electro-chemiluminescence.

Anti-TOM20 (#sc-11415, 1:200 for immunofluorescence staining) and anti-NQO1 (#sc-32793, 1:1000 for immunoblotting) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#10494-1-AP, 1:20,000 for immunoblotting) was purchased from Proteintech (Rosemont, IL, USA). Alexa Fluor 488 (#A11001) or Alexa Fluor 700 (#A21036)–conjugated secondary antibodies, Alexa Fluor 488–conjugated phalloidin (#A12379), and 4′,6-diamidino-2-phenylindole (DAPI) (#1306) were obtained from Invitrogen (Carlsbad, CA, USA). IRDye800CW-labeled anti-rabbit secondary antibody (#926-32211) was purchased from LI-COR Biosciences (Lincoln, NE, USA). Isoplumbagin (5-hydroxy-3-methyl-1,4-naphthoquinone) was acquired from AKos GmbH (Steinen, Germany). Oligomycin (#75351), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (#C2920), rotenone (#R8875), antimycin A (#A8674), lactobionic acid (#153516), taurine (#T8691), digitonin (#D141), glutamate (#G8415), malate (#M1000), succinate (#S3674), adenosine 5′-diphosphate sodium salt (ADP) (#A2754), TMPD (#T3134), and ascorbate (#A4034) were purchased from Sigma-Aldrich (St Louis, MO, USA).