Antibodies against hsp27 and gapdh

Cell lysis was performed using Triton buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM Na₃VO₄, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 1% Triton X-100, 50 mM NaF plus 1% Calbiochem Protease Inhibitor Cocktail) and lysates were assayed for protein using the Bradford protein assay, then diluted with 5× Laemmli loading buffer for SDS-PAGE. Equal amounts of protein were loaded in 4–20% Tris/glycine gels, and electrophoresed for 120 min at 130 Volts constant voltage. The gel was blotted onto a PVDF membrane by electrophoretic transfer at 25 V constant voltage overnight. The membrane was washed, blocked with 5% milk, and probed with primary antibodies. Appropriate secondary antibodies conjugated to horseradish peroxidase (Pierce, Rockford, IL) and a chemiluminescent substrate (SuperSignal, Pierce, Rockford, IL) were used to visualize immunoreactive bands. The primary antibodies against pHsp27, pPKD, and COX-2 were purchased from Cell Signaling (Danvers, MA), antibodies against Hsp27 and GAPDH were purchased from Santa Cruz (Santa Cruz, CA). Donkey anti-mouse and donkey anti-rabbit secondary antibodies were purchased from Pierce (Rockford, IL). Protein expression was analyzed with software Image J and was normalized relative to total protein for each lane (“relative expression”).

Cells were lysed in Triton buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM Na₃VO₄, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 1% Triton X-100, 50 mM NaF plus 1% Calbiochem Protease Inhibitor Cocktail) and lysates were assayed for protein using the Bradford protein assay and then diluted with 5× Laemmli loading buffer for SDS-PAGE. Equal amounts of protein were then loaded in 4–20% Tris/glycine gels, and electrophoresed for 120 min at 130 Volts constant voltage. Next, the gel was blotted onto a PVDF membrane by electrophoretic transfer at 25 V constant voltage overnight. The membrane was then washed, blocked with 5% milk, and probed with primary antibodies. Appropriate secondary antibodies conjugated to horseradish peroxidase (Pierce, Rockford, IL) and a chemiluminescent substrate (SuperSignal, Pierce, Rockford, IL) were used to visualize immunoreactive bands. The primary antibodies against pHSP27 and COX2 were purchased from Cell Signaling (Danvers, MA), antibodies against HSP27 and GAPDH were purchased from Santa Cruz (Santa Cruz, CA). Donkey anti-mouse and donkey anti-rabbit secondary antibodies were purchased from Pierce (Rockford, IL).