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. 2013 Mar 26;8(3):e59842. doi: 10.1371/journal.pone.0059842

Figure 5. Silencing of the BLK kinase leads to alteration of the association between BANK1 and PLCg2.

Figure 5

(A) Immunoblot of extracts derived from human Daudi B-cells showing efficient silencing of endogenous BLK protein. The cell line shBLK was transduced with lentivirus coding for small hairpin RNAs targeting the BLK kinase while shControl lentivirus codes for unrelated sequences. Top, western blot analysis using antibody to BLK; bottom, western blot analysis using antibody to GAPDH as loading control. (B) The relative BLK mRNA is reduced to half in the silencing line (shBLK)(***P<0,001 based on Student´s test comparing mRNA expression between Daudi cell versus cBLK silencing cells and control silenced cells versus BLK silencing cells). (C) Immunoprecipitates (IPs) of stimulated silenced shBLK cells with anti-IgM, using anti-PLCg2 antibody and interrogated with anti-BANK1 to assay BANK1-PLCg2 association. Quantification of the immunoprecipitates normalized with BANK1 is displayed below the bands. Silencing of BLK leads to less association between BANK1-PLCg2. (D) Kinetics of the association between BANK1-PLCg2 assayed with in situ Proximity Ligation (PLA) in control and BLK-silenced cell lines. The association between BANK1-PLCg2 is reduced in silenced cells during resting conditions and after 15 minutes of IgM stimulation (***P≤0,001 based on Student´s test comparing control cells versus BLK silencing cells). The difference of BANK1-PLCg2 association between control and the silenced lines was not significant at 1 minute after stimulation.