Chemidoc mp detection system

Western blots were performed on cell extracts from HT29. Protein concentrations of extracts were determined by Uptima CooAssay Max Protein Assay Kit (Interchim, UP87542A) to ensure equal loading. Proteins were separated by electrophoresis on 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for HSPB1 detection and 10% SDS-PAGE for HSPA1A detection. HSPB1 (StressMarq, SPR-105B) and HSPA1A (StressMarq, SPR-103B) human recombinant proteins were used as standard controls. Separated proteins were transferred onto nitrocellulose membranes using a Bio-Rad Trans-Blot Turbo Transfer System. HSPB1 was detected using a monoclonal HSPB1 antibody (StressMarq, SMC-161A) at 1:5000 dilution as the primary antibody and anti-mouse IgG–peroxidase (Sigma-Aldrich, A5278) at 1:2000 as the secondary antibody. HSPA1A was detected by HSPA1A-DEG-EI (in house) diluted at 1:2000 as the primary antibody and ExtraAvidin®-Peroxidase (Sigma-Aldrich, E2886) diluted 1:5000 as secondary antibody. Membranes were washed with TBS-Tween 20 and then incubated for 5 min in SuperSignal West Femto Maximum Sensitivity (Thermo Scientific) substrate. Chemiluminescence was detected using the ChemiDoc MP detection system (Bio-Rad, Hercules, CA, USA).

Co-immunoprecipitation was carried out using HeLa cells expressing HA-tagged GJA1-20k. Cytoskeletal-protein interactions were stabilized by the addition of phalloidin (25 μM, Sigma-Aldrich) during cell lysis in 0.5% NP40 buffer (in mM, 150 KCl, 20 HEPES, 2 MgCl₂, 2 K₂HPO₄, 1 DTT, 1 NaF, 0.1 Na₃VO₄, 0.5% NP40, pH7.4 with halt protease inhibitor). Cells were rotated for 30 min and spun down at 10,000 × g for 20 min at 4°C to remove insoluble debris. Following protein normalization, cell lysates were precleared using Dynabeads protein G (Thermo Fisher Scientific) for 30 min with rotation at 4°C. Beads were discarded, and 2 mg of precleared lysate was used for each reaction. Immunoprecipitation was undertaken using 5 μg of either mouse anti-HA (4C12, Abcam), or mouse anti-GST (B-14, Santa-Cruz Biotechnology) as negative isotype control at 4°C for 4 h with rotation. Dynabeads protein G (20 μl) was added to each reaction, and the tubes were rotated for an additional 45 min at room temperature. Protein complexes were washed 3 times with lysis buffer using a Dynamag-2 magnet. Proteins were then eluted with 30 μl 2X NuPAGE sample buffer containing 100 mM DTT, incubated at 37° for 20 min, and subjected to SDS-PAGE electrophoresis and Western blotting. Membranes were imaged using the ChemiDoc MP detection system (Bio-Rad). The experiments were repeated three times.

Total protein from GCs and TCs were extracted using M-PER^® mammalian protein extraction reagent according to the manufacturer’s instructions and protein levels were quantified using the Pierce™ BCA Protein Assay Kit. Halt™ Protease and Phosphatase Inhibitor Cocktails were added to the samples final solution to avoid protein degradation. Samples (20–40 μg) were resolved on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to Hybond-P PVDF membrane (GE Amersham, Amersham, UK). Membranes were then probed at 4 °C overnight in 5% BSA in TTBS with different primary antibodies (details and dilutions for each antibody are indicated in Table 1). After washing three times with TTBS, membranes were incubated for 1 h at room temperature with anti-rabbit HRP-conjugated IgG diluted in 5% non-fat dry milk in TTBS. Protein bands were visualized by chemiluminescence (ECL; Millipore, Burlington, MA, USA) and quantified using a ChemiDoc MP detection system (Bio-Rad) and Image Lab™ software version 6.0.1.

Western blot analyses were carried out on selected proteins (CLU, VTN, CRYBB2 and CRYBB3) to confirm the proteomic data. 25 μg of proteins from each sample was separated by 4–12% SDS-PAGE and transferred to PVDF membrane. Membranes were blocked by Tris-buffered saline containing 5% milk for 1 hour, and primary antibodies added at a final concentration of 1–2 μg/ml. After incubation with the primary antibody overnight at 4 °C, blots were washed with Tris-buffered saline and then incubated with anti-IgG antibody linked to horseradish peroxidase for 2 hours. Signals indicative of protein levels were detected using an enhanced chemiluminescent substrate (Clarity Western ECL Substrate, Bio Rad) and Bio-Rad ChemiDoc MP detection system and band intensities quantified in the linear range of detection.

Total protein from GC was extracted using M-PER^® mammalian protein extraction reagent according to the manufacturer’s instructions and protein levels were quantified using the Pierce™ BCA Protein Assay Kit. Halt™ Protease and Phosphatase Inhibitor Cocktails were added to the samples’ final solutions to avoid protein degradation. Samples (20–40 μg) were resolved on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to Hybond-P PVDF membrane (GE Amersham, Mississauga, ON, Canada). Membranes were then probed at 4 °C overnight in 5% BSA in TTBS with different primary antibodies (details and dilutions for each antibody are indicated in Table 1). After washing three times with TTBS, membranes were incubated for 1 h at room temperature with anti-rabbit HRP-conjugated IgG diluted in 5% non-fat dry milk in TTBS. Protein bands were visualized by chemiluminescence (ECL; Millipore, Billerica, MA, USA) and quantified using a ChemiDoc MP detection system (Bio-Rad, Hercules, CA, USA) and Image Lab™ software.

After ZEA exposure, cells were washed with cold PBS and lysed in SDS loading buffer (50 mM Tris-HCl, pH6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM ethylene diamine tetra-acetic acid and 0.02% bromophenol blue). Samples were separated via 10% SDS–PAGE and blotted onto Hybond-P PVDF membrane (GE Amersham, Piscataway, NJ, USA). After transfer, the membranes were blocked in TTBS-BSA (10 mM Tris–HCl, 150 mM NaCl, 0.1% Tween-20, 5% BSA, pH 7.5) for 1 h at room temperature. Membranes were incubated overnight at 4 °C with the primary antibody against mitogen-activated protein kinase 8, 14 and 3/1 (MAPK8, MAPK14, MAPK1/3) (# 9252, 9215, 9102) and their phosphorylated form (#9251, 9211, 9101) (Cell Signaling Technology, Danvers, MA, USA) diluted 1:1000 in TTBS-BSA. After three washing steps with TTBS, membranes were incubated for 1 h at 25 °C with 1:10,000 anti-rabbit HRP-conjugated IgG (W401B) (Promega, Madison, WI, USA) diluted in TTBS-5% non-fat dry milk. After three washes in TTBS, protein bands were visualized by chemiluminescence using Immobilon western chemiluminescent HRP substrate (Millipore, Etobicoke, ON, Canada) and quantified using a ChemiDoc MP detection system and Image Lab™ software (Bio-Rad, Mississauga, ON, Canada).