Fig. 2.
Immunoprecipitation and immunoblots of wild-type (WT) and Pank1−/− Syn-Pank2−/− (dKO) brains. Forebrain lysates were prepared from dKO and age-matched WT animals at P19-P20. Numbered lanes 1-3 contain immunoprecipitates from WT brains (n=3), and numbered lanes 4-6 contain immunoprecipitates from dKO brains (n=3). A. PanK proteins were immunoprecipitated from the lysates using a pan-reactive anti-PanK antibody, fractionated by SDS-PAGE and immunoblotted with the same anti-PANK antibody as described in Materials and methods. The far-left lane contains lysates from HEK 293T cells that overexpressed mouse PanK1α (61 kDa), PanK2 (48 kDa) and PanK3 (41 kDa) proteins as indicated and the 2nd lane from left contains purified recombinant mouse PanK1β (41 kDa). Pank1β and PanK3 protein standards have the same molecular size and were loaded onto gels separately. The 3rd lane from left contains Protein A-Sepharose beads alone without lysate. Non-specific immunereactive band (ns) detected at 55 kDa (immunoglobin) are indicated with an asterisk (*). B. Immunoblot with anti-PanK2 antibody following the identical immunoprecipitation described in panel A of WT and dKO forebrain lysates age P19-P20. C. The brain lysates (20 μg protein per lane) that corresponded to the numbered immunoprecipitates in panels A and B stained with Coomassie Brilliant Blue (CBB) to indicate equivalent protein input in the immunoprecipitation assay. D. Quantification of the PanK2 protein in the dKO forebrains levels as % of WT forebrains shown in panel B. The immunoprecipitation-immunoblot assay was performed independently with brain lysates from 6 mice of each genotype and the data represent the mean ± SEM. Statistical significance values (shown in red) was calculated using the parametric t-test comparing the band intensities of the WT and the dKO.