Anti glyceraldehyde 3 phosphate dehydrogenase gapdh

The primary antibodies were anti-ANGPTL7 (Abcam, Cambridge, UK), anti-RohA (CST, Beverly, MA, USA), anti-p-MLC (CST), anti-MLC (CST), anti-SP1 (Abcam), phalloidin-488 (Cytoskeleton, Denver, CO, USA), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam). Y27632, a ROCK inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA). Dexamethasone (Sigma D4902) was purchased from Sigma-Aldrich.

Anti-DUSP5 and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were obtained from Abcam (Cambridge, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Antibodies directed against CHOP, spliced form of X-box-binding protein-1 (XBP1s), IRE1, and ATF6, phosphorylated ERK, ERK or cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-linked anti-rabbit and anti-mouse IgGs were also provided from Cell Signaling Technology. TM, ISRIB, and other reagents were supplied from Sigma (St. Louis, MO, USA).

Total protein was extracted from H9c2 cells and MSCs-exosomes by
radio-immunoprecipitation assay (RIPA) lysis buffer (TaKaRa, Japan) with
protease inhibitors (Roche, China). The concentrations of proteins were detected
by a bicinchoninic acid (BCA) protein assay kit (Pierce, Netherlands). Sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to
separate equal quantities of total proteins (20 μg per lane), and then separated
proteins were transferred onto polyvinylidene difluoride membrane (Millipore,
MA, USA). Membranes were blocked by PBS-5% fat-free dried milk at room
temperature for 1 h and then incubated at 4 ℃ overnight with anti-CD63
(1:1,000), anti-YAP (1:2,000), anti-phosphor (p)-YAP (1:2,000), anti-tafazzin
(TAZ) (1:2,000), anti-caspase 3 (1:1,000), anti-B-cell lymphoma-2 (Bcl-2)
(1:2,000), anti-Bax (1:1,000), and anti- glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (1:3,000) (Abcam, UK). Then, the goat anti-rabbit
horseradish peroxidase-conjugated secondary antibody was incubated with
membranes for 2 h. Protein bands were visualized by ChemiDoc XRS+ system
(Bio-Rad, CA, USA).

Spleen samples were lysed in RIPA buffer containing Halt protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific, San Diego, CA, USA) and incubated for 45 min at 4 °C. Samples of lysate equivalent to 20 μg of protein were separated by 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and proteins were transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with Bovine Serum Albumin (Gibco, Carlsbad, CA, USA) and incubated with the following primary antibodies; anti-MCL1 (Proteintech, Chicago, IL, USA), anti-BCL2 (Bioss Biotechnology, Beijing, China), anti-BAX (Bioss Biotechnology), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam, Cambridge, UK) for 15 h at 4 °C. The membranes were washed again and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG, goat anti-mouse IgG (both Thermo Fisher Scientific, San Diego, CA, USA), or mouse anti-goat IgG (Santa Cruz Biotechnology, Dallas, TX, USA). Bound proteins were visualized using SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific, San Diego, CA, USA), and the membranes were analyzed using an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).

Samples were prepared as described previously [95 (link)] and analyzed with 4–12% and 12% precast SDS-PAGE gels (no. NP0321, NP0322 and NP0342, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Levels of scanned films (no. 28906837, GE Healthcare, Buckinghamshire, UK) were adjusted in Adobe Photoshop CS4 Extended in accordance with the guidelines for the proper handling of digital image data given in [96 (link)].
The following primary antibodies were used: anti-extracellular signal-regulated kinase 1/2 (ERK1/2) (no. 9102, Cell Signaling, Danvers, MA, USA; a kind gift from Leonard Girnita and Claire Worrall, Karolinska Institutet, Stockholm, Sweden), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; no. ab8245, Abcam, Cambridge, UK), anti-NAD(P)H:quinone oxidoreductase 1 (NQO1; no. sc-32793, Santa Cruz, Heidelberg, Germany), anti-Nrf2; no. ab62352, Abcam), anti-p21 (118) [97 (link)], anti-p53 (DO-7) [98 (link)], anti-phospho-ERK1/2 (no. 9101, Cell Signaling; a kind gift from Leonard Girnita and Claire Worrall) and anti-phospho-histone H2AX (Ser 139) (γ-H2AX; no. 05-636, Millipore, Molsheim Cedex, France). Horseradish peroxidase (HRP)-conjugated secondary antibodies were rabbit anti-mouse (no. P0161, Dako, Glostrup, Denmark) and swine anti-rabbit (no. P0211, Dako).