Suppression of DLBCL Progression by the E3 Ligase Trim35 Is Mediated by CLOCK Degradation and NK Cell Infiltration

Figure 3

Trim35 controls the ubiquitination and degradation of CLOCK. (a) 293FT cells were transfected with Flag-tagged Trim35 or an empty vector. Forty-eight hours after transfection, cell lysates were prepared and subjected to IP with anti-Flag beads and immunoblotting as indicated. (b) Immunoblotting of lysed 293FT cells transfected with Trim35 or empty vector, along with HA-Ub. Cell lysates were prepared and subjected to IP with anti-HA beads and immunoblotting as indicated. (c) Trim35 sgRNAs are designed to target the human Trim35 loci. Agarose gel shows modification at both loci in transfected cells. (d) Pfeiffer cells were infected with viruses expressing Trim35 sgRNA or a control sgRNA and selected; cell lysates were immunoblotted for the indicated proteins. Densitometry quantified protein bands, presented as relative expression. (e) Pfeiffer cells were infected with Flag-Trim35 expression virus. Protein extracts were immunoblotted for the indicated proteins. CLOCK bands were quantified by densitometry and presented as relative expression. Pfeiffer cells with stable Trim35 overexpression (f) and Trim35-depleted Pfeiffer cells (g) were treated with a proteasome inhibitor MG132 (10 μg/mL) during the last 5 hours before lysis. CLOCK protein levels were detected and analyzed. Protein bands were quantified by densitometry and presented as relative expression. (h, i) The same cells as described in (e, d) were used for qRT-PCR analysis of CLOCK mRNA expression. Results are normalized to GAPDH mRNA level and expressed fold changes in mRNA expression compared with control. (j, k) The same cells as described in (e, d) were cultured for 6 h and 24 h before being further incubated with cycloheximide (CHX) for the indicated time points. The level of CLOCK at different time points was detected by western blot. Densitometry quantified CLOCK bands, presented as relative expression. Each experiment was successfully carried out three times. ^NS, , , and .

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