Human/Mouse/Rat Phospho-GSK-3 alpha / beta (S21/S9) Antibody

Detection of Human Phospho-GSK‑3 alpha (S21) and GSK-3 beta (S9) by Western Blot. Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line untreated (-) or treated (+) with 200 nM PMA for 20 minutes and MCF-7 human breast cancer cell line untreated or treated with 100 ng/mL Recombinant Human IGF-1 (291-G1) for 15 minutes. PVDF membrane was probed with 0.2 µg/mL of Human/Mouse/Rat Phospho-GSK-3a/ beta (S21/S9) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1590), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (HAF008). Specific bands were detected for Phospho-GSK-3a (S21) and GSK-3 beta (S9) at approximately 51 and 46 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Phospho-GSK‑3 alpha / beta (S21/S9) in HeLa Human Cell Line. Phospho-GSK‑3 alpha / beta (S21/S9) was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line treated with PMA (left panel; positive staining) and untreated HeLa cell line (right panel; negative control) using Rabbit Anti-Human/Mouse/Rat Phospho-GSK‑3 alpha / beta (S21/S9) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1590) at 8 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red; NL004) and counterstained with DAPI (blue). Specific staining was localized to nuclei. Staining was performed using our protocol for Fluorescent ICC Staining of Non-adherent Cells.

Detection of Rat GSK-3 alpha/beta by Western Blot HYS-32 modulates GSK3 beta phosphorylation.(A) Cell lysates from control astrocytes (Con) or astrocytes treated for 2, 6, 12, or 24 h with 5 μM HYS-32 were subjected to 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GSK3 beta -pTyr216, GSK3 beta -pSer9, or GAPDH. (B) Densitometric analyses of GSK3 beta -pTyr216 and GSK3 beta -pSer9 expressed as the density of the bands in the treated groups relative to the control. *p<0.05 compared to control using one-way ANOVA with Dunnett’s post-hoc test. The results were collected from five independent experiments. (C) According to the data in (B), the stacked bar graph showing the relative percentage of phospho-GSK3 beta at indicated time. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0126217), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Rat GSK-3 alpha/beta by Western Blot LY294002 inhibits the HYS-32-induced phosphorylation of GSK3 beta -pS9 and GSK3 beta -pY216.(A) Control astrocytes (Con) or astrocytes treated for 24 h with 5 μM HYS-32 (HYS), co-treated for 24 h with 5 μM HYS-32 and 20 μM LY294002 (HYS+LY), or treated with 20 μM LY294002 (LY) were subjected to 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GSK3 beta -pSer9, GSK3 beta -pY216, totalGSK3 beta, or GAPDH. (B) Densitometric analyses of GSK3 beta -pSer9 and GSK3 beta -pY216 expressed as the density of the bands in the treated groups relative to the control. (C) According to the data in (B), the stacked bar graph showing relative percentage ofphospho-GSK3 beta in various treatment. (D)Astrocytes treated as in (A) were fixed in cold acetone and triple-stained for beta -tubulin, N-cadherin, and F-actin. Quantitative analysis of the straight distance between microtubule tips and cell border were performed as described in Materials and Methods. The results were collected from three independent experiments.*p<0.01 compared to HYS using one-way ANOVA with Dunnett’s post-hoctest. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0126217), licensed under a CC-BY license. Not internally tested by R&D Systems.