Fig. 5: Identification of TBK1 as a key mediator contributing to BBOX1 function in ccRCC.
From: BBOX1 restrains TBK1-mTORC1 oncogenic signaling in clear cell renal cell carcinoma
a IP-mass spectrometry (MS) from 786-O and Caki-1 tumor lysates identified TBK1 as a top candidate protein interacting with BBOX1. b List of top BBOX1 binding proteins identified by IP-MS from 786-O and Caki-1 xenograft tumors. The full list of MS-identified proteins is provided in Supplementary Data 1. c Co-immunoprecipitation of endogenous TBK1 and endogenous BBOX1 in A498 xenograft tumors. d Co-immunoprecipitation of endogenous TBK1 and HA-tagged wild type BBOX1 or catalytic dead (CD) mutant in 786-O xenograft tumors or in vitro cells. e–g Immunoblots (e), ECAR measurement (n = 5 independent cell cultures, The median value (center line), lower quartile and upper quartile (box edges) and maximum and minimum value whiskers are indicated in the boxplot.) (f), and soft agar growth (n = 3 biological cell cultures) (g) of A498 cells expressing indicated BBOX1 sgRNA and TBK1 shRNA grown in HPLM medium. Each experiment has been repeated at least two times with similar results. Scale bar, 1 mm. h, i Subcutaneous xenograft tumor growth (h) and tumor weight (i) of A498 cells expressing indicated BBOX1 sgRNA and TBK1 shRNA. n = 8 mice for each group. Immunoblots (j) and soft agar growth (n = 3 biological cell cultures) (k) of A498 cells expressing indicated BBOX1 expression vector and TBK1 shRNA grown in HPLM medium. Each experiment has been repeated at least two times with similar results. Scale bar, 1 mm. Subcutaneous xenograft tumor growth (l), and tumor weight (m) of A498 cells expressing indicated BBOX1 expression vector and TBK1 shRNA. n = 6 mice for each group. Statistical analysis was conducted by two-way ANOVA followed by Tukey’s multiple comparison test (h and l) or two-tailed Student’s t test (f, g, i, k, and m). Error bars represent SEM, NS denotes no significance. Representative immunoblots and gels shown in figures were repeated at least two times independently with similar results. Source data are provided as a Source Data file.
