Hsp70

Protein lysates were prepared from cultured cells or frozen tumors using a 9M Urea, 0.075 M Tris buffer (pH 7.6) and quantified using the Bradford assay. 50-100μg of protein was subjected to reducing SDS/PAGE by standard methods. Western blots were incubated with primary antibodies against AXL (R&D Systems), P-AXL (Cell Signaling), GAS-6 (R&D), AKT (Cell Signaling), and phospho-AKT (Cell Signaling) as needed. To confirm equal protein loading, blots were probed with antibodies specific for β-actin (Sigma Aldrich), HSP70 (Thermo Fisher), or Vinculin (BD Pharmingen).

Western blot analysis was performed as previously described [5 (link)]. The following antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA); phosphorylated Akt (Ser⁴⁷³, 1:500, #9271, Rabbit polyclonal), phosphorylated Erk (Thr²⁰²/Tyr²⁰⁴, 1:1000, #4377, Rabbit monoclonal), phosphorylated Src (Tyr⁴¹⁶, 1:100, #2101, Rabbit polyclonal), Akt (1:2000, #9272, Rabbit polyclonal), Erk (1:2000, #9102, Rabbit polyclonal) and Src (1:1000, #2109, Rabbit polyclonal). Hsp70 (1:500.000, #MS-482, Mouse monoclonal) were purchased from Thermo Scientific and β-actin (1:500.000, #A5441, Mouse monoclonal) from Sigma Aldrich. The experiments were performed at least twice and representative blots are shown.

Denatured protein samples from whole cell lysates and anti-ChAT IP/co-IPs were resolved on 7.5–15% SDS-PAGE gels, then transferred to PVDF membranes (Bio-Rad). For immunoblotting, membranes were blocked for 1 h at room temperature in 5% non-fat milk powder in PBS containing 0.15% Triton X-100 (PBST), followed by incubation overnight at 4°C with primary antibody. Probed membranes were washed with PBST, and primary antibodies were detected using 1:10,000 peroxidase-coupled secondary antibodies (Jackson ImmunoResearch) and Clarity Western ECL Substrate (Bio-Rad) on a ChemiDoc MP system (Bio-Rad). The following primary antibodies were used: 1:1,000 ChAT (CTab) (Dobransky et al., 2003 (link)), 1:10,000 β-actin (Sigma), 1:10,000 GAPDH (Cell Signalling), 1:1,000 HSC70 (StressMarq), 1:2,000 HSP70 (Thermo), 1:2,000 HSP90 (StressMarq), 1:1,000 CHIP (Santa Cruz), 1:1,000 ubiquitin (Santa Cruz), 1:1,000 FLAG-M2 (Sigma), and 1:1,000 LC3B-I/II (Thermo). For anti-CHIP immunoblotting from anti-ChAT co-IP samples, 1:1,000 VeriBlot IP secondary antibody (Abcam) was used to reduce IgG light chain interference from detecting endogenous 35-kDa CHIP.

Cell extracts were prepared in lysis buffer (containing 0.5 M Tris, 2.5 M NaCl, 500 mM NaF and 10% nonionic P₄₀ with protease inhibitors: 200 mM Na₃VO₄, 0.5 M β-glycerophosphate, 0.25 M NaPPi, 0.1 M PMSF, 1 mg/mL leupeptine, 0.1 M benzamidine and 1 mg/mL aprotinin). Protein concentration was measured using protein assay dye reagent (Bio-Rad) according to the manufacturer’s instructions. For Western blotting analysis, 40 μg total protein lysates were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore) and blocked with either 5% nonfat milk in PBS-T or 3% BSA in TBS-T. The membrane was then probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibody and developed with ECL-detecting reagents (Thermo Scientific) and autoradiography film. The band intensity was quantified using Scion Image (Scion Corporation). The following primary antibodies were used: phospho-IKK, phospho-IκB, phosphor-STAT1, STAT1, Mcl-1, Bcl-2, Bcl-xL, caspase-3 (Cell Signaling), HSP70 (Thermo Scientific), HSF-1 (Stressgen), NFκB p65, p50 (Santa Cruz Biotechnology) and HIF-1α (Novus Biologicals).

Caco-2 cells pretreated with different ALA concentrations for 24 h and exposed to HS for 6 h (HSF1, Nrf2) or 24 h (HSP70, E-cadherin) were lysed using 50 µl RIPA lysis buffer (Thermo Scientific, Rockford, IL, USA) containing protease inhibitors (Roche Applied Science, Penzberg, Germany). Lysates were normalized for protein content and WB analysis was conducted as described previously [17 (link)] using primary antibodies against HSF1 (1:1000; Cell Signaling, Danvers, MA, USA), HSP70 (1:1000; Enzo Life Sciences, Farmingdale, NY, USA) or E-cadherin (1:1000, eBioscience, San Diego, CA, USA), and β-actin antibody (1:4000; Cell Signaling) for equality of sample loading. For the detection of Nrf2, the nuclear protein extracts were separated using NE-PER Nuclear and Cytoplasmic Extraction Reagents based on the manufacturer’s instructions (Pierce, Rockford, IL, USA). Nuclear protein concentrations were measured, normalized, and the WB analysis was conducted using primary antibodies against Nrf2 (1:1000; Cell Signaling) and Lamin A (1:1000; Cell Signaling) for equality of loading. Digital images were obtained with ChemiDoc™ MP imager (Bio-Rad Laboratories Inc.) and signal intensities were quantified using the ImageJ 1.47 software.
The protein expression was normalized with β-actin or Lamin A (for nuclear proteins) and expressed as mean fold change in relation to the control group.