Fig 1: RASD2 activates MAPK signaling by promoting RAF1 (Ser338) phosphorylation. (A) Co‐IP followed by LC–MS identified RAF1 as a RASD2‐binding protein. (B) Co‐IP and western blot assays confirmed the RASD2‐RAF1 interaction in 769‐P cells. (C) Kaplan–Meier survival curves for 102 ccRCC patients stratified by RAF1 expression (high vs. low) over 60 months. (D, E) Western blot analysis of RAF1 and p‐RAF1 (Ser338) levels in RASD2‐KO 769‐P cells (C) and RASD2‐OE Caki‐2 cells (D). n = 3. (F) The correlation analysis in GEPIA2 shows that RASD2 and RAF1 expression levels are weakly positively correlated. (G) Western blot analysis confirmed the successful generation of RAF1‐knockout (RAF1‐KO) 769‐P cells using CRISPR/Cas9. These cells were subsequently transduced with lentiviruses encoding either wild‐type RAF1 (RAF1‐WT) or a serine‐to‐alanine point mutation at position 338 (RAF1‐S338A). n = 3. (H) Transwell assays were performed to assess migration and invasion in 769‐P cells under the following conditions: RAF1‐KO, RAF1‐KO reconstituted with RAF1‐WT, and RAF1‐KO expressing RAF1‐S388A. (I) Transwell assays evaluated migration and invasion in Caki‐2 cells with or without RASD2OE after BAY43‐9006 treatment. n = 4. (J) Western blot analysis of MAPK signaling in Caki‐2 cells with or without RASD2OE following BAY43‐9006 treatment for 12 h. n = 3. Values are mean ± SEM. Statistical analysis: (C) log‐rank test, (D, E) unpaired two‐tailed t‐test; (G–J) one‐way ANOVA followed by a post hoc test for multiple comparisons.
Fig 2: RASD2 promotes tumorigenicity in nude mice. (A) Caki‐2 cells with or without RASD2OE were inoculated into nude mice flanks. Once tumors reached 3–5 mm in diameter, mice were treated with BAY43‐9006 (5 mg/kg daily for 10–12 days). Images show xenograft tumors isolated 21 days postinoculation, with tumor weight quantification. n = 5. (B) Western blot analysis of p‐RAF1, RAF1, and RASD2 expression in Caki‐2 cells with or without RASD2OE following BAY43‐9006 treatment. n = 3. (C) Immunohistochemistry analysis of RASD2, Ki67, RAF1, and p‐RAF1 expression in Caki‐2 cells with or without RASD2OE after BAY43‐9006 treatment. n = 4. (D) Immunohistochemistry analysis of E‐Cadherin, N‐Cadherin, and Vimentin expression in Caki‐2 cells with or without RASD2OE after BAY43‐9006 treatment. Values are mean ± SEM. Statistical analysis: All p values by one‐way ANOVA test followed by a post hoc test for multiple comparisons.
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