Anti hsp70

Anti-β-actin was from Santa Cruz Biotechnology (sc-47778); anti-Hsp70 from Enzo Life Sciences (catalog no. ADI-SPA-810); anti-Hsc70 from StressMarq Biosciences Inc (catalog no. SMC-151) or anti-Hsp70 (BB70, Dr. David Toft, Mayo Clinic, Rochester, MN); anti-Hsp90β (H90.10, Dr. David Toft); anti-LC3B (Cell Signaling Technology, 3868S);, anti-cleaved LC3A (LC3AII) (Abgent Antibody, Abcepta, San Diego, CA, AP1805a); anti-GAPDH (Santa Cruz Biotechnology, sc-166545); and p62/SQSTM1 from Santa Cruz Biotechnology, Inc (catalog no. sc-28359).

Eye tissue sections was incubated in 4% goat serum containing anti-myocilin (Santa Cruz, Dallas, TX, 1:50, # sc-21243) or anti-Hsp70 (Enzo Life Sciences, Farmingdale, NY, 1:100,# ADI-SPA-810-D) primary antibodies overnight. The following day, sections underwent PBS washing, and tissue was incubated with Alexa Fluor 594 (Invitrogen, Grand Island, NY; 1:500) secondary antibody for 2 hours and washed in PBS. Sections were then stained with DAPI for 20 minutes, washed and cover-slipped using ProLong Gold Antifade Reagent (Invitrogen).

Cell lysates were prepared in M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) with Mini protease and Halt phosphatase inhibitor cocktails (Thermo Scientific) and protein concentration was determined using the BCA assay (Thermo Scientific). 25 μg of total cellular protein was separated on SDS/PAGE gels and transferred to nitrocellulose membrane that was blocked for 1 hour at room temperature in 5% nonfat milk. Primary antibodies were added overnight at 4°C with gentle shaking. The primary antibodies used were: anti-TP53 (Sigma), anti-CDKN1A (Cell Signaling), anti-NRF2 (Abcam), anti-KEAP1 (Abcam), anti-ubiquitin (Cell Signaling), anti-HSP70 (Enzo), anti-HSP90 (Cell Signaling), anti-pAKTThr308 (Cell Signaling), anti-pAKTSer473 (Cell Signaling) and anti-AKT (Cell Signaling). After incubation with the appropriate secondary antibody, signals were detected using ECL Western Blotting Substrate (Thermo Scientific). Equal protein loading in each lane was confirmed with β-actin antibody (Sigma). Densitometry analysis was performed using the freely available image-processing program ImageJ (NIH).

For Western blotting, skeletal muscle tissues were homogenized with RIPA buffer. After the lysates were centrifuged at 13,000 rpm for 30 min, the supernatants were measured using bicinchoninic acid (BCA) (Thermo Fisher Scientific, Waltham, MA, USA) assay and boiled for 5 min at 100 °C after mixing with a 5× sample buffer. Western blotting samples were loaded onto SDS-PAGE gels, run, and transferred onto PVDF membranes (Merck Millipore, Burlington, MA, USA). After blocking with Tris Buffered Saline with Tween 20 (TBST) containing 5% skim milk, the membranes were incubated with primary antibodies overnight at 4 °C and then incubated with secondary antibodies for 1–2 h at RT. The membranes were incubated with ECL solution (Promega, Madison, WI, USA) and imaged using ImageQuant LAS 500 (GE Healthcare, Chicago, IL, USA).
The primary antibodies used were anti-BIS [1 (link)], anti-HSF1, anti-HSP70 (Enzo Life Sciences), anti-GAPDH, anti-HSPB5, anti-YAP1 (Santa Cruz Biotechnology), anti-cleaved PARP1, anti-desmin, anti-HSPB8, anti-p62 (ABCAm), anti-COX4 (Cell Signaling Technology, Danvers, MA, USA), and anti-filamin C (Novus Biologicals, Centennial, CO, USA).

Detection of the protein expression in the ventricular myocardium by Western blot was performed as described previously [23 (link)]. The protein concentration of the supernatant was measured by the BCA kit (Thermo Scientific, Waltham, MA, USA). Twenty micrograms of protein extract was separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to a nitrocellulose membrane. After blocking in 5% bovine serum albumin for 1 hour, blots were then incubated overnight at 4°C with the following primary antibodies: anti-NF-κB p-p65, anti-p65, anti-cleaved caspase 3, anti-LC3B, anti-Atg5-12, anti-p62, anti-p-mTOR, and anti-mTOR (all 1 : 1000, Cell Signaling Technology, Danvers, MA, USA); anti-TNF-α (1 : 1000, Abcam, Cambridge, MA, USA); anti-Hsp70 (1 : 1000, Enzo Life Sciences, Farmingdale, NY, USA); anti-α-actin (1 : 5000, GeneTex, Irvine, CA, USA). The membranes were then probed with horseradish peroxidase-conjugated secondary antibodies. Proteins were visualized using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA) and quantified by densitometry of the blots using ImageJ software.