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. Author manuscript; available in PMC: 2014 Dec 23.
Published in final edited form as: Dev Cell. 2013 Dec 23;27(6):607–620. doi: 10.1016/j.devcel.2013.11.013

Figure 4. Involvement of Calpain 2 in Megakaryocytic Downregulation of MePCE and Consequences of MePCE Downregulation.

Figure 4

(A) Effects of calpain 2 knockdown on MePCE (Me), HEXIM1 (H1) and LARP7 (L7) levels. Cells transduced and cultured as in Figure 3B underwent immunoblotting followed by scanning densitometry. Graph depicts mean ± SEM for three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.005; NS, not significant.

(B) In vitro analysis of MePCE cleavage by calpain 2. Extracts from HEK293T cells transfected with FLAG-MePCE (FL-Me) expression vector were incubated with 50 to 200ng purified calpain 2/S1 ± 2 mM CaCl2 (Ca2+) followed by immunoblot for FL-Me and GAPDH.

(C) In vitro analysis of MePCE cleavage by calpain 2. Purified FL-Me protein from HEK293T transfectants was subjected to in vitro cleavage and immunoblot as in (B).

(D–E) K562 cells transduced with shRNA constructs targeting MePCE underwent immunoblotting in (D). Cell morphology was assessed by light microscopy on Wright-stained cytospins (200X) in (E).

(F) Effect of MePCE downregulation on P-TEFb interaction with HEXIM1. Extracts from K562 cells subjected to shRNA knockdown of MePCE underwent immunoprecipitation of cyclin T1 followed by immunoblotting for HEXIM1 (H1) and cyclin T1 (CT1) See also Figure S3.