Ab75745

Immunohistochemistry was performed as previously described [16 (link)]. Briefly, sections containing auditory cortex were double-immunostained with rabbit anti-GSK3α (1:200, G08-63R-25, SignalChem, USA), rabbit anti-GSK3β (1:1000, ab32391, Abcam, USA), or rabbit GSK3β phospho-Y216 (1:150, ab75745 Abcam, USA), and mouse anti-PV (1:5000, 235, Swant, Switzerland). Images of the auditory cortex were captured using confocal microscope (Nikon A1). NIH ImageJ software was used to measure the integrated density of GSK3α, GSK3β and GSK3-phospho (Y279/Y216) in PV-positive interneurons or non-PV neurons. The corrected total cell fluorescence (CTCF) was calculated as previously described [26 (link)]. Results are presented as normalized florescence, which is the ratio of CTCF value of the protein to the CTCF values of corresponding proteins in non-PV neurons of floxed-control mice.

Total protein was extracted from cultured neurons and the ischemic penumbra of the rat cortex using cell lysis buffer supplemented with proteinase and phosphatase inhibitors. The nuclear proteins were extracted using a commercial kit (Beyotime, China). Cell lysates were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked in 5% non-fat milk TBST buffer for 1.5 h at room temperature. The membranes were incubated in primary antibody overnight at 4 °C and in secondary antibody for 1 h at room temperature. Dilutions for primary antibodies were as follows: anti-GSK-3β (#9315, 1:1000, Cell Signaling Technology, Boston, MA, USA), anti-β-catenin (#9582, 1:1000, Cell Signaling Technology), anti-Nrf2 (YT3189, 1:500, Immunoway, Houston, TX, USA), anti-GSK-3β (phospho-tyr216) (ab75745, 1:500, Abcam, Cambridge, MA, USA), anti-LaminB1 (ab133741, 1:500, Abcam), anti-HO-1 (BS6626, 1:500, Bioworld, St. Louis Park, Minnesota, USA), anti-NQO1 (BS6833, 1:500, Bioworld), and anti-actin (BS 6007 M, 1:10,000, Bioworld).The secondary antibody was diluted 1:5000 (Sangon Biotech, S hanghai, Co., Ltd.). The density of bands was detected using an imaging densitometer (Bio-Rad, Foster City, CA, USA), and the gray value of bands was quantified using Quantity One 1-D analysis software.

Western blot analysis was performed following our previous description [28 (link)]. Antibodies against LMNB1 (1: 1000, Santa Cruz, sc-377000), Snail (1: 1000, CST, # 3879), MMP11 (1: 1000, Abcam, ab119284), Slug (1: 1000, CST, # 9585), N-cadherin (1: 1000, CST, # 13116), Akt (1: 1000, CST, # 4691), GSK-3β (1: 2000, Abcam, ab93926), Phospho-Akt (p-Akt) (1: 2000, CST, #4060), Phospho-GSK-3β (p-GSK-3β) (1: 1000, Abcam, ab75745) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1: 10000, Abcam, ab8245) were used for this assay. GAPDH was selected as the internal control. Finally, an electrochemiluminescence imaging analysis system was used to detect the signals of the bands.

Total protein was isolated using lysis buffer (Cell Signaling Technology) containing protease inhibitors (Pierce Biotechnology, Waltham, USA). Equal amounts of protein were loaded on a 10% SDS-PAGE gel. The polyvinylidene fluoride membranes were blotted with the following primary antibodies, including mouse anti-E-cadherin (Abcam, ab76055), mouse anti-ICAM1 (Abcam, ab2213), mouse anti-vimentin (Abcam, ab8978), rabbit anti-p-GSK3 β (Abcam, ab75745), rabbit anti-β-catenin (Abcam, ab16051), rabbit anti-wnt3a (Abcam, ab234099) and Mouse anti-GAPDH (Multi sciences, ab011-040).
After 1 h incubating with secondary antibodies, anti-Mouse IgG-HRP (Thermos, RA230188) and anti-Rabbit IgG-HRP (ab205718), the blots were visualized by the Alphalmager™ 2000 Imaging System (Alpha Innotech, San Leandro, USA). The band density was quantified using ImageJ.