Journal: bioRxiv

Article Title: Active Gαi/o mutants accelerate breast tumor metastasis via the c-Src pathway

doi: 10.1101/2023.01.16.524334

Figure Lengend Snippet: A-B , representative immunoblots showing the expression of Gα i2 R179C and Gα oA R243H in MCF10A cells ( A ) and activation of c-Src and STAT3 by phosphorylation of Src Y416 and STAT3 Y705 in MCF10A cells treated with vehicle control (CT), 2 μM of saracatinib (sara) and STAT3-IN-1 (STAT3i) (B) . CD , the effect of Gα i2 R179C and Gα oA R243H expression on the growth of MCF10A cells in 2D culture ( C ) and in Matrigel ( D ). E , representative images showing the absence of tumor formation in nude mice orthotopically implanted with MCF10A cells expressing GFP, Gα i2 R179C or Gα oA R243H. *, **,*** p<0.05, 0.01 and 0.001 vs. GFP, n=4-6. One-way ANOVA was used for statistical analysis in this Figure.

Article Snippet: Antibodies for EGFR (no. 2232), phospho-EGFR Y1068 (no. 3777), EGFR (no. 4267), HER2 (no. 2165), phospho-HER2 Y1221/1222 (no. 2243), AKT (no. 4685), phospho-AKT S473 (no. 4060), Src (no. 2109), phospho-Src Y416 (no. 6943), ERK1/2 (no. 4696) and phospho-ERK1/2 T202/Y204 (no. 4370) were obtained from Cell Signaling Technology; GADPH (sc-47724) from Santa Cruz Biotechnology; mouse anti-ee antibody (no. 901801) from Biolegend.

Techniques: Western Blot, Expressing, Activation Assay

Journal: bioRxiv

Article Title: Active Gαi/o mutants accelerate breast tumor metastasis via the c-Src pathway

doi: 10.1101/2023.01.16.524334

Figure Lengend Snippet: A-B , Western blotting ( A ) showing increased phosphorylation of c-Src Y416 and EGFR Y1068 and no change in phosphorylation of AKT s473 , STAT3 Y705 and HER2 Y1221/1222 in Neu/Gα i2 R179C tumors as compared to Neu tumors. Each lane represents a sample from an individual tumor. The Western blotting data from A were quantified and expressed as the ratio of the phosphorylated to total proteins ( B ). Two tail unpaired Student’s t test was used for statistical analysis of the data in B , and p values are shown. C-D , Western blotting showing phosphorylation of the indicated proteins in Neu cells expressing GFP, Gα i2 R179C (Gα i2 ) or Gα oA R243H (Gα oA ) and treated with vehicle control (CT) or saracatinib (sara; 2 μM) ( C ) and Neu cells overexpressing GFP or the constitutively active c-Src Y527F mutant ( D) . E , co-immunoprecipitation of ee-tagged Gα i2 R179C and Gα oA R243H with c-Src in Neu cells. Neu cells expressing GFP, Gα i2 R179C (Gα i2 ) or Gα oA R243H (Gα oA ) were immunoprecipitated with the anti-ee antibody and probed with the indicated antibodies.

Article Snippet: Antibodies for EGFR (no. 2232), phospho-EGFR Y1068 (no. 3777), EGFR (no. 4267), HER2 (no. 2165), phospho-HER2 Y1221/1222 (no. 2243), AKT (no. 4685), phospho-AKT S473 (no. 4060), Src (no. 2109), phospho-Src Y416 (no. 6943), ERK1/2 (no. 4696) and phospho-ERK1/2 T202/Y204 (no. 4370) were obtained from Cell Signaling Technology; GADPH (sc-47724) from Santa Cruz Biotechnology; mouse anti-ee antibody (no. 901801) from Biolegend.

Techniques: Western Blot, Expressing, Mutagenesis, Immunoprecipitation