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. 2023 Feb 9;11(2):e004871. doi: 10.1136/jitc-2022-004871

Figure 6.

Figure 6

CAMB reduces PDL1 through GSK3β/β-TrCP ubiquitination promotion. (A) RT-qPCR for CD274 mRNA in OVCAR5 and ID8agg cells treated with (+) or without (−) chlorambucil (10 µM for OVCAR5 and 2.5 µM for ID8agg) for 48 hours. (n=4). (B) Western blot for PDL1 protein in OVCAR5 cells with (+) or without (−) MG132 (2 µM) for 6 or 20 hours (upper panel). PDL1 expression in OVCAR5 cells treated with chlorambucil (2.5 µM) 48 hours with or without MG132 (2 µM) in the final 6 hours (lower panel). (C) OVCAR5 cells treated with chlorambucil for indicated times (hours) and then PDL1 was immunoprecipitated (IP) using anti-PDL1 (1:50) antibody and subjected to Western blot analysis with anti-ubiquitin (Ub) antibody. Molecular weights shown to left of IP blot. (D) Western blot for PDL1 after 48-hour incubation of OVCAR5 cells with chlorambucil (2.5 µM) incubation, siRNA against β-TrCP (si) or both. (E) Western blot for GSK3β activation and β-TrCP in OVCAR5 cells after chlorambucil (2.5 µM, 48 hours) incubation. (F) Western blot for PDL1 with chlorambucil (2.5 µM) or epidermal growth factor (EGF) 30 ng/mL for 48 hours in OVCAR5 cells. CAMB, chlorambucil; VC, vehicle control.