Semi dry electroblotter

Total proteins were extracted from 1 g of 14-day-old seedlings and dissolved in sample buffer (50 mM Na₂HPO₄/NaH₂PO₄, pH 7.4; 150 mM NaCl; 1% Triton X-100; 15% glycerol; 1 mM PMSF; and 1 × cocktail). Isolated proteins were identified using 10% sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. They were transferred onto polyvinylidene difluoride (PVDF) membranes using a semi-dry electroblotter (Bio-Rad). The PVDF membranes were probed with anti-FLAG (Sigma), anti-H3 (Agrisera), anti-LFR (Lin et al., 2018 (link)), anti-SWI3B (Sarnowski et al., 2002 (link)) or anti-tubulin antibody (Sigma). Goat anti-rabbit or anti-mouse IgG secondary antibodies were used for immunodetection.

Infected HFFs were passed through a 27G needle and syringe to release intact parasites, then host cell debris was filtered away and parasites were collected by centrifugation. Parasites were resuspended in RIPA buffer (50 mM Tris-HCl pH 7.4,150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40 substitute, 0.1% SDS) to prepare protein lysates. SDS sample buffer was added to protein lysates, which were then subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membranes using a Semi-Dry Electroblotter (BioRad). The antioxidant proteins were detected using a rabbit α-HA primary antibody (Cell Signaling). Primary antibodies were detected by an α-rabbit IgG secondary antibody conjugated to horseradish peroxidase (Cell Signaling). Membranes were incubated with SuperSignal West Femto Chemiluminescent substrate (Pierce) for 5 minutes then imaged using a FluorChem E system machine (Protein Simple).

From cytosolic extracts of CEM-3R43 or CEM-C7-14 cells, 60μl were prepared buffer (0.01 M Tris, 0.0015 mM EDTA, 0.5 mM DTT, 1x protease inhibitor cocktail, pH 7.4) with or without 3 M TMAO. In another set, cytosolic extracts were prepared from cells treated with 50 mM TMAO as described in whole cell GR:GC binding assay (above). Cytosols were divided equally and incubated under “non-activating” and “activating” conditions, while maintaining the TMAO concentration. The preparations then had 5 μl of GR antibody (Affinity Bioreagents, Golden, CO) added, followed by incubation for an additional 1 hour at 4°C. Each sample was raised to a final volume of 0.5 ml, and 25 μl of protein agarose conjugate were added, followed by incubation for a further 2 hours. Pellets were collected by centrifugation, and washed thoroughly. The beads were resuspended in SDS-sample buffer and boiled for 5 min. After SDS-PAGE gel electrophoresis, the proteins were transferred onto a PVDF membrane (Bio-Rad) using a semi-dry electroblotter (Bio-Rad) and immunoblotted using an antibodies raised against HSP90 (AC88) (Santa Cruz Biotechnology, Santa Cruz, CA), or the other chaperones indicated (kind gift of Dr. David Toft).

An SDS-page was performed using 25 μg of total protein loaded into a 10% polyacrylamide gel. After electrophoresis, proteins were transferred to a nitrocellulose membrane using a semi-dry electroblotter (Bio-Rad). The membrane was processed for chemiluminescence detection using Luminata Forte Western HRP Substrate (Millipore) according to the manufacturer's instructions. The primary antibodies were: rabbit anti-TRPM8 (Ab109308, Abcam, detecting the pore region of TRPM8), rabbit anti-HA tag (Sc-805, Santa Cruz) and goat anti-GA3PDH (Sc-20357, Santa Cruz).