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. 2020 Feb 27;1:10–22. doi: 10.1016/j.crimmu.2020.02.001

Fig. 5.

Fig. 5

Validation of CIP2A protein interaction by IP-Western Blot and PLA Confocal microscopy. (A) CIP2A IP performed in Th17 ​cells and WB detection to validate the CIP2A known interaction with phosphatase PP2A-A. Total lysate (Input) and control IP (IgG) and CIP2A IP using Ab2 are shown on the blot. A conformational specific secondary antibody was used to probe proteins without interference from the denatured IgG heavy (50 kDa) and light chains (25 kDa). (B) WB validation of the CIP2A-PPP1CA interaction. CIP2A immunoprecipitated with two different antibodies (Ab1 and Ab2) in Th17 ​cells (72h) and detected as in A. (C-D) Proximity ligation assay (PLA) assay validation of the CIP2A interaction with PPP1CA (C) and TRIM21 and UBR5 (D) in Th17 ​cells (D). The GFP antibody was used as a negative control in the PLA. DAPI was used to stain the nuclei. Scale bar is 7μm. Representation of the interaction between CIP2A-PPP1CA (C) or TRIM21 and UBR5 with CIP2A (D) by PLA as a univariate dot plot. The average number of PLA signals (spots) per cell (n ​= ​10 images from a representative of three experiments) was plotted. Statistics by paired two tailed T-test, where ∗ represents P ​< ​0.05. and ∗∗< 0.01. Scale bar is 7μm. (E) Western blot validations representative of interactions between TRIM21, IRF4, STAT1 and PP2A with CIP2A in Th17 ​cells at 72h. IP was performed using Ab1 and Ab2 CIP2A antibodies and a conformational specific secondary antibody was used to detect the protein as in Fig. 5A. A representative Western blot of four biological replicates is shown, the remaining three Western blot replicates are shown in Figs. S5B–D. (F) CIP2A inhibition leads to enhanced Nf-κB activation. CIP2A is silenced in three different individual donor cell pools, and CIP2A, phospho (pp52 and pp110) and total (p52 and pp110) Nf-κB2 and Actin are shown on the blot. The bar chart on the right shows the quantification of the phosphorylation levels of p110 and p52 in CIP2A silenced Th17 ​cells. Quantification was performed using ImageJ software in which phosphorylation values were normalised with respective total protein p110 and p52 expression. Statistical significance calculated using Student's t-test (two-tailed paired) where ∗ denotes p-value <0.05.