MOESM1 of Ras isoforms: signaling specificities in CD40 pathway

Additional file 1. Figure S1. (A) Densitometry for immunoblot analysis for the silencing of Ras isoforms H, K, and N-Ras using specific siRNA. (B-D) Densitometry for immunoblots for phosphorylation of p38MAPK and ERK1/2 in P388D1 cells silenced for H (B), K (C), and N-Ras (D). Figure S2. (A) Densitometric analysis of the activation of H-Ras, K-Ras, and N-Ras on the silencing of Ras GEFs (Sos-1/2, Vav, and Ras-GRP) using GEF specific siRNA. (B) Densitometry of immunoblot analysis of phosphorylation of p38MAPK and ERK1/2 on silencing of Ras GEFs Sos-1/2, Vav and Ras-GRP. Figure S3. (A) Densitometric quantifications of the blots in Figure 5A. (B)Densitometric analyses of immunoblots of activated Ras isoforms in the lysates of untransfected or Syk or Lyn specific siRNA transfected, anti-CD40 antibody (3μg/ml) treated P388D1 cells, normalized to corresponding controls. (C) Densitometric analyses of immunoblots of translocated Sos-1/2 (Tr-Sos-1/2), translocated Ras-GRP (Tr-RasGRP), syk and lyn in the lysates of untreated or Syk siRNA or Lyn siRNA or anti-CD40 antibody (3μg/ml) treated P388D1 cells, normalized to corresponding controls. (D) Densitometric analyses of immunoblots of translocated Sos-1/2 (Tr-Sos-1/2), translocated Ras-GRP (Tr-Ras-GRP), phospho-lyn (p-lyn) and phospho-syk (p-syk) in the lysates of untreated or Syk inhibitor (Syk Inh, 3μM; Calbiochem, San Diego, CA) or PP-1 (340nM; BIOMOL International, PA) treated or anti-CD40 antibody (3μg/ml) treated macrophages, normalized to corresponding controls. (E) Co-immunoprecipitation of H-Ras, K-Ras and N-Ras at different doses of anti-CD40 to check for its association with Lyn, Syk and CD40. Figure S4. (A)Densitometry for effect of silencing of H-Ras, K-Ras, and N-Ras on the phosphorylation of PI3K and Raf. Text 1. Sequence and Structure Similarity Among Ras isoforms. Table S1. Sequence and structure similarity among Ras isoforms. Text 2. Comparative studies on symmetry of residue-residue interaction preferences, across Ras isoform structures. Table S2. Quantifying the symmetry in Residue-Residue interaction in three Ras isoforms.

附加文件1 补充图S1。（A）使用特异性小干扰RNA（siRNA）沉默Ras同工型H-Ras、K-Ras及N-Ras的免疫印迹光密度分析。（B-D）分别在沉默H-Ras（B）、K-Ras（C）、N-Ras（D）的P388D1细胞中，针对p38丝裂原活化蛋白激酶（p38MAPK）与细胞外调节蛋白激酶1/2（ERK1/2）磷酸化水平的免疫印迹光密度分析。补充图S2。（A）使用针对Ras鸟苷酸交换因子（Ras GEFs，包括Sos-1/2、Vav及Ras-GRP）的特异性siRNA沉默后，对H-Ras、K-Ras及N-Ras活化状态的光密度分析；（B）沉默Ras GEFs Sos-1/2、Vav及Ras-GRP后，针对p38MAPK与ERK1/2磷酸化水平的免疫印迹光密度分析。补充图S3。（A）对图5A中免疫印迹条带的光密度定量分析；（B）未转染、或转染Syk或Lyn特异性siRNA、或经抗CD40抗体（3μg/ml）处理的P388D1细胞裂解液中活化型Ras同工型的免疫印迹光密度分析，结果均归一化至相应对照组；（C）未处理、或经Syk siRNA、Lyn siRNA、或抗CD40抗体（3μg/ml）处理的P388D1细胞裂解液中，转位Sos-1/2（Tr-Sos-1/2）、转位Ras-GRP（Tr-RasGRP）、Syk及Lyn的免疫印迹光密度分析，结果归一化至相应对照组；（D）未处理、或经Syk抑制剂（Syk Inh，3μM；Calbiochem，美国加利福尼亚州圣地亚哥）、PP-1（340nM；BIOMOL International，宾夕法尼亚州）处理、或经抗CD40抗体（3μg/ml）处理的巨噬细胞裂解液中，转位Sos-1/2（Tr-Sos-1/2）、转位Ras-GRP（Tr-Ras-GRP）、磷酸化Lyn（p-Lyn）及磷酸化Syk（p-Syk）的免疫印迹光密度分析，结果归一化至相应对照组；（E）不同剂量抗CD40处理下的H-Ras、K-Ras及N-Ras免疫共沉淀实验，以检测其与Lyn、Syk及CD40的结合情况。补充图S4。（A）沉默H-Ras、K-Ras及N-Ras对磷脂酰肌醇3-激酶（PI3K）与Raf激酶磷酸化水平影响的光密度分析。文本1：Ras同工型的序列与结构相似性。附表S1：Ras同工型间的序列与结构相似性。文本2：跨Ras同工型结构的残基-残基相互作用偏好对称性比较研究。附表S2：三种Ras同工型中残基-残基相互作用对称性的定量分析。