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. 2020 Mar 30;122(11):1673–1685. doi: 10.1038/s41416-020-0779-9

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© The Author(s), under exclusive licence to Cancer Research UK 2020

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Fig. 2 — a DLD-1 cells were transfected with Flag-CSN6 plasmids. The protein level of TRIM21 was immunoblotted with anti-TRIM21 antibodies. b DLD-1 cells were infected with scrambled or CSN6-specific shRNA lentivirus. The protein level of TRIM21 was immunoblotted with anti-TRIM21 antibodies. c Quantitative RT-PCR analysis was performed to measure the mRNA levels of Trim21 in cells transfected with empty vector or Flag-CSN6. d Flag-TRIM21 was expressed in HCT116 cells. Flag-TRIM21 was immunoprecipitated with anti-Flag, and the associated CSN6 was detected by western blotting. e HEK293T cell lysates were immunoprecipitated with an anti-TRIM21 antibody and immunoblotted with anti-TRIM21 and anti-CSN6 antibodies. f HEK293T cells were co-transfected with Flag-TRIM21 and the HA-CSN6-WT, CSN6-N terminal or CSN6-C terminal construct. Cell lysates were immunoprecipitated with anti-Flag and subsequently immunoblotted with anti-Flag and anti-HA antibodies. Heavy heavy immunoglobulin chain, Light light immunoglobulin chain. g HEK293T cells were co-transfected with HA-CSN6 and the Flag-TRIM21-WT, TRIM21-N terminal or TRIM21-C terminal construct. Cell lysates were immunoprecipitated with anti-Flag and subsequently immunoblotted with anti-Flag and anti-HA antibodies. h Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells carrying scrambled or TRIM21-specific siRNA. Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells carrying empty vector or Flag-TRIM21 plasmids. i Sphere-formation assay of DLD-1 cells transduced with control or Flag-TRIM21 lentivirus. j HCT116 cells were transfected with Flag-CSN6 plasmids. The protein level of TRIM21, OCT1 and ALDH1A1 was immunoblotted with anti-TRIM21, anti-OCT1 and anti-ALDH1A1 antibodies. k Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells with TRIM21 knockout. l Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells stably expressing empty vector or Flag-CSN6 and rescued with Flag-TRIM21 plasmids. m Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells stably expressing scrambled or CSN6-specific shRNA and rescued with TRIM21-specific siRNA. n The knockout efficiency of TRIM21 in HCT116 cells by TRIM21 sgRNAs was evaluated by western blotting. Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in control or HCT116 cells with TRIM21 knockout and then transfected with empty vector or Flag-CSN6. *P < 0.05, **P < 0.01 and ***P < 0.001.