Figure 6
Figure 6. SPRY2 downregulate MAPK-ERK signaling by interacting with RAF-1, Syk, and BRAF. (A) Mec-1 cells were transfected with pLKO-TET-empty and pLKO-TET-Spry2. Cells were treated with DOX for 4 days to induce the expression of SPRY2. Displayed is a scanned western blot of CLL cells transfected with empty and spry2 cDNA to measure the protein levels of SPRY2 and p-Erk upon DOX treatment. β-actin and PAN (total) Erk1/2 were used as controls. (B) KRAS-V12 and BRAF-V600 mutants were co-transfected with empty and Spry2 vectors in Mec-1 cells. Scanned western blot showing the p-Erk and Erk levels in these cells (left). Scanned western blot of SPRY2 and BRAF protein levels in the same cells (right); β-actin was used as loading control. (C) Human nB cells were isolated from healthy donors and cells were stimulated by BCR crosslinking for 24 hours. Cells were lysed to prepare lysate for immunoprecipitation (IP) (described in supplemental Methods). Displayed is a scanned western blot of immunoprecipitation with SPRY2 and Syk showing pull-down of RAF-1, Syk, BRAF, and SPRY2. IgG, Pi3K, and BTK were used as negative controls. (D) nB cells and Mec-1 cells were stimulated as described in (C) and cyotospin were prepared using these cells. Cells were stained with SPRY2, p-Syk, and DAPI fluorescence antibody. Displayed are immunofluorescence images showing co-localization of SPRY2 and p-Syk in nB cells and Mec-1 CLL cells. Unstimulated cells, goat-IgG, and rabbit-IgG were used as control. (E) nB cells were isolated from spleen of CD19-cre;Spry2(wt) and CD19-cre;Spry2(tg) mice, and stimulated for 30 minutes with anti-IgM antibody. Displayed is scanned western blot of p-Syk and Syk, and densitometric measurements showing normalized decreased levels of p-Syk. (F) Mec-1 CLL cells were overexpressed with Spry2 cDNA and empty vector co-expressing GFP; cytospins were prepared of these cells. Slides were stained with p-Syk (red) antibody. Displayed are immunofluorescence images and densitometric measurements showing decreased levels of normalized p-Syk in SPRY2 co-expressing GFP-positive cells. DAPI, 4,6 diamidino-2-phenylindole; IB, immunoblotting.

SPRY2 downregulate MAPK-ERK signaling by interacting with RAF-1, Syk, and BRAF. (A) Mec-1 cells were transfected with pLKO-TET-empty and pLKO-TET-Spry2. Cells were treated with DOX for 4 days to induce the expression of SPRY2. Displayed is a scanned western blot of CLL cells transfected with empty and spry2 cDNA to measure the protein levels of SPRY2 and p-Erk upon DOX treatment. β-actin and PAN (total) Erk1/2 were used as controls. (B) KRAS-V12 and BRAF-V600 mutants were co-transfected with empty and Spry2 vectors in Mec-1 cells. Scanned western blot showing the p-Erk and Erk levels in these cells (left). Scanned western blot of SPRY2 and BRAF protein levels in the same cells (right); β-actin was used as loading control. (C) Human nB cells were isolated from healthy donors and cells were stimulated by BCR crosslinking for 24 hours. Cells were lysed to prepare lysate for immunoprecipitation (IP) (described in supplemental Methods). Displayed is a scanned western blot of immunoprecipitation with SPRY2 and Syk showing pull-down of RAF-1, Syk, BRAF, and SPRY2. IgG, Pi3K, and BTK were used as negative controls. (D) nB cells and Mec-1 cells were stimulated as described in (C) and cyotospin were prepared using these cells. Cells were stained with SPRY2, p-Syk, and DAPI fluorescence antibody. Displayed are immunofluorescence images showing co-localization of SPRY2 and p-Syk in nB cells and Mec-1 CLL cells. Unstimulated cells, goat-IgG, and rabbit-IgG were used as control. (E) nB cells were isolated from spleen of CD19-cre;Spry2(wt) and CD19-cre;Spry2(tg) mice, and stimulated for 30 minutes with anti-IgM antibody. Displayed is scanned western blot of p-Syk and Syk, and densitometric measurements showing normalized decreased levels of p-Syk. (F) Mec-1 CLL cells were overexpressed with Spry2 cDNA and empty vector co-expressing GFP; cytospins were prepared of these cells. Slides were stained with p-Syk (red) antibody. Displayed are immunofluorescence images and densitometric measurements showing decreased levels of normalized p-Syk in SPRY2 co-expressing GFP-positive cells. DAPI, 4,6 diamidino-2-phenylindole; IB, immunoblotting.

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