Gpr30

Following treatment, the cells cultured on chamber slides were fixed with ice-cold 100% methanol for 10 min, and incubated with primary antibody against vimentin (1:1000, IgG Corp, Nashville, TN, USA), GPR30 (1:1000, Bioss. Woburn, MA, USA), or Ki-67 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. Cells were washed with PBS and then incubated with secondary IgG antibodies conjugated to Alexa 488 or 555 (1:500, Invitrogen, Carlsbad, CA, USA) for 45 min at room temperature. The nuclei were counterstained with TP-PRO-3. Excess antibody was removed by three washes and cells were imaged with a Zeiss LSM-510 confocal microscope.

Muscle homogenates were separated by SDS-PAGE and transferred onto membranes, as previously described [13 (link)]. Immunoblots were probed by antibodies against GPR30 (1:500, Bioss, Woburn, MA, USA), heat shock protein (HSP) 90 (1:1000, BD Biosciences, San Jose CA, USA), HSP70 (1:1000, BD Biosciences), HSP27 (1:1000, Abcam, Inc., Cambridge, MA, USA), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000; Cell Signaling, Danvers, MA, USA) was used as a loading control.

Cultured cells or LV tissue homogenates from mRen2.Lewis rats were separated by SDS-PAGE and transferred onto membranes, as previously described [10 (link),11 (link)]. Immunoblots were probed using antibodies for GPR30 (1:500, Bioss, Inc.), chymase (1μg/ml, Bioss, Inc.), and CDK1 (1:1000, Abcam, Cambridge, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5,000; Cell Signaling, Danvers, MA, USA) was used as a loading control.