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. 2023 Apr 24;24(9):7782. doi: 10.3390/ijms24097782

Figure 4.

Figure 4

TRIM21 controls basal levels of PDGFRβ in growing cells but does not affect receptor mRNA levels. (a,b) Depletion of TRIM21 increases basal levels of PDGFRβ but not the rate of degradation after stimulation with PDGF-BB. After transient silencing of TRIM21 in fully growing (a) or serum-starved cells for 16 h (b), U2OS cells were pretreated with cycloheximide (CHX) for 1 h and stimulated with PDGF-BB for the indicated time periods. Expression levels of PDGFRβ, TRIM21 and α-tubulin, as a loading control, were determined by immunoblotting (Ib) of total cell lysates (TCL) (c,d). Overexpression of TRIM21 decreases the total levels of PDGFRβ but does not affect its stability of upon ligand stimulation. The experiment was performed using the tet-inducible U2OS-TRIM21 cell line, that were growing in full media (c) or serum-starved for 16 h (d), treated with cycloheximide and stimulated with PDGF-BB as described in (a). TCL was subjected to immunoblotting for PDGFRβ, MYC-epitope to detect MYC-tagged TRIM21 and α-tubulin. (e,f) mRNA expression levels of PDGFRβ do not change upon knockdown of TRIM21. Total RNA was extracted from U2OS cells that were treated with control or TRIM21 siRNA for 72 h. mRNA was reverse transcribed and amplified to detect expression levels of PDGFRβ (e) or TRIM21 (f). Expression was normalized against the control gene HPRT, the average values for four independent repeats are presented in the graph. ** p < 0.01; “ns”—non-significant.