Hsp40

The following antibodies were used for western blotting: p53 (OP43; Merck Millipore), MDM2 (SMP14) (sc-965; Santa Cruz), AKT (2920; Cell Signaling Technology), phospho-AKT (Thr308) (9275; Cell Signaling Technology), phospho-AKT (Ser473) (9271; Cell Signaling Technology), PARP (9532; Cell Signaling Technology), caspase 3 (9661; Cell Signaling Technology), phospho-5′ AMP-activated protein kinase ([AMPK] bs-4002R; Bioss), AMPK (E-AB-30490; Elabscience Biotechnology Inc.), phospho-mammalian target of rapamycin ([mTOR] (E-AB-20929; Elabscience Biotechnology Inc.), mTOR (E-AB-32129; Elabscience Biotechnology Inc.), LC3A/B (4108; Cell Signaling Technology), α-tubulin (NB100-690; Novus Biologicals), GAPDH (10494-1-AP; Proteintech), HSP-40 (sc-59554; Santa Cruz Biotechnology), CHIP (sc-133066; Santa Cruz Biotechnology), and vinculin (GTX109749; GeneTex). For co-immunoprecipitation assay, cell lysates were incubated with p53 antibody (OP43, EMD Millipore, Billerica, MA) or HSP-40 antibody (sc-398766, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. Then samples were precipitated with appropriate protein A/G beads with matched IgG as negative control. The precipitants were analysed in SDS-PAGE western blot.

Protein expression was determined by immunoblot analysis as previously described (34 (link)). Antibodies used were sourced as follows: anti-HSP70-1 (C92F3A-5) and -HSF-1 were from Enzo Life Sciences, Inc. (Farmingdale, NY); anti-HSP70-1 (MA3009) was from Thermo Fisher Scientific; anti-td-Tomato was from MyBioSource, Inc. (San Diego, CA); and anti-p53, -HSP90, -HSP40, -CD9, -CD63, -LAMP-2, and -β-actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-HSP70 IgG ELISA kit from Enzo Life Sciences, Inc., and the TeloTAGGG^TM Telomerase PCR ELISA from Roche (Basel, Switzerland) were used according to the manufacturer's instructions. RPMI 8226 cells were treated overnight with RTX or IVIgG (both at 5 mg/ml), and the culture supernatant was collected. Cells were then washed in PBS to remove these antibodies, lysed in IP Lysis buffer (Thermo Fisher Scientific), and both the harvested supernatant and the cell lysate was incubated with protein A/G agarose (Sigma-Aldrich). The agarose beads were incubated overnight at 4°C and washed in IP Lysis buffer and immunoblotting was performed for HSP70-1.