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. 2020 Apr 13;14(6):1282–1296. doi: 10.1002/1878-0261.12676

Fig. 5.

Fig. 5

LNCAROD forms complex with HSPA1A and YBX1 and increases YBX1 protein stability in a HSPA1A‐dependent manner. (A) Schematic diagram of identification of LNCAROD binding proteins by RNA pull‐down coupled with mass spectrometry. (B) RNA pull‐down and western blot indicated that LNCAROD binds with HSPA1A and YBX1 proteins. (C) RIP‐qPCR assays demonstrated that HSPA1A and YBX1 bind with LNCAROD. (D) Subcellular fractionation and western blot assays indicated that YBX1 and HSPA1A proteins distribute in cytoplasm and nucleus in HK1 and FaDu cells. (E) Association of HSPA1A and YBX1 proteins with deletion fragments of LNCAROD were assessed by western blot. (F) Co‐IP assays suggested that both exogenous and endogenous YBX1 proteins were co‐immunoprecipitated with HSPA1A in HK1 cells. (G) Co‐IP assay revealed that RNase A treatment with cell lysate weakened YBX1‐HSPA1A association in HK1 cell. (H) YBX1‐HSPA1A association was assessed by Co‐IP assays in LNCAROD stably silenced HK1 cell and LNCAROD stably overexpressed Tca8113 cell. (I) RNA level of LNCAROD was measured in cells upon transient or stable loss of YBX1 (n = 3 per group). (J) RNA levels of HSPA1A and LNCAROD were measured by RT‐PCR upon transient silence of HSPA1A in HK1 cell (n = 3 per group). (K) HSPA1A and YBX1 protein levels in LNCAROD‐depleted cells were determined by western blot assays. (L) mRNAs levels of YBX1 and HSPAIA in HK1 and FaDu cells upon loss of LNCAROD were determined by RT‐qPCR assays (n = 3 per group). (M) HSPA1A and YBX1 protein levels were measured by western blot assays in Tca8113 cells with overexpression of LNCAROD. (N) HSPA1A and YBX1 mRNA levels in Tca8113 cell were measured by RT‐qPCR assays (n = 3 per group). (O) Pulse‐chase assay of YBX1 protein levels in HK1/sh‐LNCAROD cells treated with CHX. (P) MG132 treatment prevented reduction of YBX1 protein levels in HK1 and FaDu upon depletion of LNCAROD. (Q) YBX1 protein levels in HK1 cells transfected with HSPA1A specific siRNAs were measured by western blot assays. (R) YBX1 mRNA levels in HK1 cells transfected with HSPA1A specific siRNAs were measured by RT‐qPCR assays (n = 3 per group). (S) Western blot assays demonstrated that MG132 treatment prevented YBX1 protein degradation in HK1cell upon loss of HSPA1A. (T) YBX1 protein levels in Tca8113 cells were measured by western blot assays. All data are mean ± SD. Data were analyzed by using Student's t‐test. **P < 0.01.