Cell culture pellets
were lysed using radioimmunoprecipitation assay buffer (Pierce, cat
no. 89901) that was supplemented with protease and phosphatase inhibitors
(Roche) and sonicated using a Bioruptor UCD-200 (Diagenode). Sonicated
lysates were centrifuged at 14,000 rpm for 30 min at 4 °C, and
supernatants were collected. Samples were estimated for protein concentration
(DC protein assay; Bio-Rad) and boiled in 6× Laemmli buffer (330
mM Tris-HCl, pH 6.8; 330 mM sodium dodecyl sulfate; 6% β-ME;
170 μM bromophenol blue; and 30% glycerol). Samples were then
loaded on gradient Mini-PROTEAN TGX precast protein gels (Bio-Rad)
and transferred to polyvinylidene fluoride membranes (Trans-Blot Turbo
transfer packs, Bio-Rad).
For protein expression analysis of
FOSL1 and FOSL2, the following Ab were used: anti-FOSL1 (Cell Signaling
Tech., cat no. 5281), anti-FOSL2 (Cell Signaling Tech., cat no. 19967),
and anti-β-actin (Sigma-Aldrich, cat no. A5441). HRP-conjugated
anti-mouse IgG (Santa Cruz Biotechnology, cat no. sc-2005) and anti-rabbit
IgG (BD PharMingen, cat no. 554021) were used as secondary Ab.
were lysed using radioimmunoprecipitation assay buffer (Pierce, cat
no. 89901) that was supplemented with protease and phosphatase inhibitors
(Roche) and sonicated using a Bioruptor UCD-200 (Diagenode). Sonicated
lysates were centrifuged at 14,000 rpm for 30 min at 4 °C, and
supernatants were collected. Samples were estimated for protein concentration
(DC protein assay; Bio-Rad) and boiled in 6× Laemmli buffer (330
mM Tris-HCl, pH 6.8; 330 mM sodium dodecyl sulfate; 6% β-ME;
170 μM bromophenol blue; and 30% glycerol). Samples were then
loaded on gradient Mini-PROTEAN TGX precast protein gels (Bio-Rad)
and transferred to polyvinylidene fluoride membranes (Trans-Blot Turbo
transfer packs, Bio-Rad).
For protein expression analysis of
FOSL1 and FOSL2, the following Ab were used: anti-FOSL1 (Cell Signaling
Tech., cat no. 5281), anti-FOSL2 (Cell Signaling Tech., cat no. 19967),
and anti-β-actin (Sigma-Aldrich, cat no. A5441). HRP-conjugated
anti-mouse IgG (Santa Cruz Biotechnology, cat no. sc-2005) and anti-rabbit
IgG (BD PharMingen, cat no. 554021) were used as secondary Ab.