Human/Mouse/Rat GSK-3 alpha / beta Antibody

Detection of Human GSK‑3 alpha / beta by Western Blot. Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line, HT-29 human colon adenocarcinoma cell line, MDA-MB-468 and MCF-7 human breast cancer cell line. PVDF membrane was probed with 0.1 µg/mL of Rabbit Anti-Human/Mouse/ Rat GSK-3a/ beta Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2157) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). Specific bands were detected for GSK-3a/ beta at approximately 46 and 51 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

GSK‑3 alpha / beta in HeLa Human Cell Line. GSK-3a/ beta was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line using Rabbit Anti-Human/Mouse/Rat GSK-3a/ beta Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2157) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Detection of GSK‑3 alpha / beta in HeLa Human Cell Line by Flow Cytometry. HeLa human cervical epithelial carcinoma cell line was stained with Rabbit Anti-Human/Mouse/Rat GSK-3a/ beta Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2157, filled histogram) or control antibody (Catalog # AB-105-C, open histogram), followed by Allophycocyanin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0111). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin.

Detection of Mouse GSK-3 alpha/beta by Western Blot GSK3 binds to scaffold formed by histidine-rich cytoplasmic loop domains within the ZIP6-ZIP10 heteromer.(a) Domain organization of ZIP10 and ZIP6 and deletion constructs used in this work to test the hypothesis that a cytoplasmic loop connecting transmembrane (TM) domains III and IV is essential for binding to GSK3 binding. (b) Control Western blot documenting ZIP6 expression products in eluates shown in panel ‘e’. (c) The co-expression of wild-type ZIP6 and ZIP10 proteins comprising intact cytoplasmic loop domains connecting TM domains III and IV is essential for binding of GSK3. GSK3-directed Western blot analysis of cellular lysates and co-immunoprecipitation eluates from in vivo formaldehyde crosslinked cells expressing different combinations of wild-type and/or mutant ZIP6/ZIP10. Note that in addition to protein bands reflecting the expected monomeric molecular weights of GSK3 and ZIP6, the Western blots in panels ‘d’ and ‘e’ revealed the appearance of high molecular weight crosslinked bands, whose appearance depended on the application of the in vivo formaldehyde crosslinking step. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28098160), licensed under a CC-BY license. Not internally tested by R&D Systems.