Figure 3 - Interaction between KPC and p27^Kip1 and subcellular localization of KPC.

(a) Interaction between endogenous p27^Kip1 and KPC1 or KPC2 in NIH 3T3 cells. Lysates of cells expressing KPC1-1 or EGFP shRNAs were subjected to immunoprecipitation (IP), followed by immunoblot analysis (IB) with anti-KPC1, anti-KPC2, or anti-p27^Kip1. (b) Association of p27^Kip1 with the KPC1–KPC2 complex in vitro. Recombinant p27^Kip1 and KPC1–KPC2 complex were mixed, immunoprecipitated with anti-p27^Kip1, and subjected to immunoblot analysis. (c) Immunofluorescence analysis of the subcellular localization of KPC1 and KPC2. NIH 3T3 cells expressing His₆–Flag–KPC1 and HA–KPC2 were stained with anti-Flag (red), anti-HA (green) and Hoechst 33258 (blue). Scale bars, 25 m. (d) Subcellular fractionation of endogenous KPC1 and KPC2. Cytosolic (C) or nuclear (N) fractions prepared from NIH 3T3 cells either in asynchronous culture (AS) or synchronized at G0, G1, or S phase were subjected to immunoblot analysis with anti-KPC1, anti-KPC2, anti--tubulin (cytosolic marker), or anti-lamin B1 (nuclear marker). Asterisk, non-specific bands.