smad2 3 antibody sampler kit  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: Promotion of stem cell-like phenotype of lung adenocarcinoma by FAM83A via stabilization of ErbB2

doi: 10.1038/s41419-024-06853-w

Figure Lengend Snippet: A Co-immunoprecipitation (Co-IP) of serine phosphorylation and ubiquitination of ErbB2 in control or FAM83A knockdown LUAD cells incubated with MG132 for an additional 4 h, using ErbB2 as bait. B Western blot analysis of FAM83A knockdown LUAD cells transfected with scramble siRNA or siRNA specific against BTrCP, CBLC, STUB1, SMURF1, using the indicated antibodies. C Co-IP of serine phosphorylation and ubiquitination of ErbB2 in FAM83A knockdown LUAD cells transfected with scramble siRNA or siRNA specific against BTrCP, CBLC, STUB1, and SMURF1. D Western blot analysis of control or STUB1 knockdown LUAD cells with FAM83A knockdown, transfected with lentivirus containing empty, STUB1 (WT), STUB1 (H260Q) construct, using the indicated antibodies. E Co-IP of the indicated proteins in LUAD cells, using FAM83A, ErbB2, or STUB1 as bait. F DuoLink PLA analysis of the direct interaction of endogenous ErbB2 with STUB1, FAM83A with ErbB2 and FAM83A with STUB1 in LUAD cells. Representative images were provided. G , H Co-IP of the indicated proteins in LUAD cells with control or FAM83A knockdown, using STUB1 or ErbB2 as bait. I DuoLink PLA analysis of the direct interaction of endogenous ErbB2 with STUB1 in LUAD cells with control or FAM83A knockdown. Representative images were provided. Data represent means ± SD of three independent experiments; * P < 0.05; ns not significant.

Article Snippet: The primary antibodies targeted S6K1 (9202 S, 1:1000), phosphorylated S6K1 (p-S6K1, T389, 9234 S, 1:1000), phosphorylated AKT (p-AKT, T308, 13038 S, 1:1000), AKT (9272 S, 1:1000), phosphorylated AKT (p-AKT, S473, 4060 S, 1:2000), β-TrCP (4394 S, 1:1000), CBLC (2747 S, 1:1000), STUB1 (2080 S, 1:1000), SMURF1 (2174 S, 1:1000) and CD44 (37259, 1:1000) were all from Cell Signaling Technology.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Phospho-proteomics, Ubiquitin Proteomics, Control, Knockdown, Incubation, Western Blot, Transfection, Construct

Journal: Cells

Article Title: Cytokine Receptor-like Factor 1 (CRLF1) and Its Role in Osteochondral Repair

doi: 10.3390/cells13090757

Figure Lengend Snippet: Chondrogenic differentiation of human BMSCs lacking CRLF1 or overexpressing CRLF1. ( A ) Alcian blue staining of sections of BMSC pellets transfected with control siRNA (siCon) or siRNA specific for CRLF1 (siCRLF1) after 6 days in chondrogenic differentiation medium; sections of BMSC pellet transduced with empty AAV (AAV) or transduced with AAV containing CRLF1 (AAV-CRLF1) after 6 days in chondrogenic differentiation medium. Bar, 100 µm. ( B ) Phosphorylated (p-Smad) and total Smad2 and Smad3 immunoblots of lysates from BMSCs transduced with empty AAV (AAV) or from BMSCs transduced with AAV-CRLF1. Cells were treated for 60 min with transforming growth factor beta 1 (TGFβ1).

Article Snippet: After blocking with a solution of low-fat milk protein, blotted proteins were immunostained with primary antibodies specific for β-actin (#3700, Cell Signaling Technology, Danvers, MA, USA), CRLF1 (ab211438, Abcam, Boston, MA, USA) or CLC (ab154798, Abcam), total STAT3 (#9139, Cell Signaling Technology), phosphorylated STAT3 (#9145, Cell Signaling Technology), total Smad2 and 3, phosphorylated Smad 2, and phosphorylated Smad3 (SMAD2/3 Antibody Sampler Kit #12747, Cell Signaling Technology), and then peroxidase-conjugated secondary antibody (Thermo Scientific Pierce, Waltham, MA, USA).

Techniques: Staining, Transfection, Control, Transduction, Western Blot