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. 2020 Apr 19;10(8):1426–1439. doi: 10.1016/j.apsb.2020.03.008

Figure 5.

Figure 5

ZDHHC12 is a CLDN3-palmitoylaiting enzyme. (A) Identification of the ZDHHC12 palmitoyltransferase by co-immunoprecipitation screening of CLDN3 and DHHC enzymes. 293T cells stably expressing FLAG-tagged CLDN3 were transfected with HA-tagged DHHCs for 24 h. Cell lysate was submitted to anti-FLAG immunoprecipitation and immunoblotting. The specific immunoblot band of HA-tagged DHHC12 co-immunoprecipitated with FLAG-tagged CLDN3 was indicated by yellow triangle. (∗represents the heavy chain of anti-FLAG antibody). (B) In-gel fluorescence shows the S-palmitoylation level of CLDN3 WT was affected by vector, DHHC12 WT and DHHC12 C127S. Right histogram shows the quantification of the fluorescence intensity relative to CLDN3 WT. Values with error bars indicate mean ± SD of four independent replicates (n.s. indicates no statistic difference; ∗∗P < 0.01). (C) The knockdown efficiency of shRNA against human ZDHHC12 in A2780 cells was validated by real-time PCR. Values with error bars indicate mean ± SD of three independent replicates (∗∗∗P < 0.001). (D) In-gel fluorescence shows the weaken S-palmitoylation level of shZDHHC12 group compared to shCtrl group. CLDN3 expressed A2780 cells with knocking down endogenous ZDHHC12 were submitted to anti-FLAG immunoprecipitation and immunoblotting. Right histogram shows the quantification of the fluorescence intensity of shZDHHC12 group relative to shCtrl group. Values with error bars indicate mean ± SD of three independent replicates (∗P < 0.05).