Hybond p 0.45 polyvinylidene difluoride membranes

Immunoblot analysis was done as described previously (64 (link)). Briefly, confluent cells were lysed in cold lysis buffer (10 mm Tris-HCl (pH 7.5), 100 mm NaCl, 10 mm EDTA, 0.5% Triton X-100, and 0.5% sodium deoxycholate) for 10 min. Aliquots of lysates were incubated with PK for 30 min at 37 °C. A proteinase inhibitor (0.5 mm Pefabloc) was used to stop PK, and samples were directly precipitated with methanol. For samples without PK treatment, Pefabloc was added directly, and samples were precipitated with methanol. Precipitated proteins were resuspended in TNE buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, and 5 mm EDTA). Samples were run on 12.5% SDS-PAGE (10.5% gel for ER stress markers), electroblotted on Hybond P 0.45 polyvinylidene difluoride membranes (Amersham Biosciences), and analyzed by immunoblot using Luminata Western chemiluminescent HRP substrates (Millipore). The densitometric analysis of immunoblots was done using ImageJ software.

Immunoblot analysis was performed as mentioned in our previous studies (29 (link)). Briefly, protein precipitated from cell lysate, BH, or SH after NaPTA precipitation was separated on 12.5% SDS-PAGE. The electroblotting was done on Amersham Biosciences Hybond P 0.45 polyvinylidene difluoride membranes (Amersham Biosciences) and analyzed using Luminata Western Chemiluminescent HRP substrates (Millipore). ImageJ software was used to perform densitometric analysis.