Research Paper Volume 16, Issue 13 pp 10765—10783

Figure 4. CALCR stabilizes CD44 via inhibiting its ubiquitination. (A) Relative mRNA levels of several potential target genes were detected by qPCR analysis in ACHN cell with CALCR depletion. (B) Venn diagram showing the DEGs correlated with CALCR expression and shorted OS in TCGA renal clear cell carcinoma. (C) Relative mRNA expression of CD44 from TCGA renal cancer (KICH, KIRC and KIRP). (D) Relative protein levels of CALCR and CD44 in 786-O cells with CALCR deficiency were determined by western blot assays. (E) Co-IP analysis suggested the endogenous binding between CALCR and CD44. (F) Relative CD44 protein level in CALCR-depleted 786-O and ACHN cells treated with CHX was analyzed by western blot assays. (G) Relative CD44 protein level in CALCR-depleted 786-O and ACHN cells treated with MG132 was determined by western blot assays. (H) The ubiquitin of CD44 immunoprecipitated by anti-CD44 or IgG antibody was detected. GAPDH served as an internal control in qPCR and western blot assays. Results were presented as mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Abbreviations: DEGs: differentially expressed genes; OS: overall survival; TCGA: the Cancer Genome Atlas; CD44: cluster of differentiation; KICH: kidney chromophobe; KIRC: kidney renal clear cell carcinoma; KIRP: kidney renal papillary cell carcinoma; CHX: Cycloheximide.