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Figure 3 | BMC Molecular Biology

Figure 3

From: Identification of two Amino Acids in the C-terminal Domain of Mouse CRY2 Essential for PER2 Interaction

Figure 3

Analysis of the interaction between mCRY2 C-terminal truncated forms with mBMAL1 using a mammalian two-hybrid system. (A) cDNA of mCry2 was cloned into pACT, where it is was fused with the VP16 domain through their N-termini. (B) HEK 293T cells were transfected with pBIND-mPer2 and pACT-mCry2-T1/T2/T3/T4/T5 along with the pGL5luc reporter plasmid. Top Panel: Expression of HA-mCRY2 and FLAG-mPER2 analyzed by western blot. Bottom Panel: Reporter activity was examined 24 h after transfection. Relative activities were calculated based on mock samples and values were plotted. Error bars indicate SEM from at least 3 experiments. * p < 0.01, ** p < 0.005, *** p < 0.0001 as determined by student's t-test compared to the control. (C) mCRY2-T4/mPER2 and mCRY2-T5/mPER2 interaction in mammalian cells. HEK293T cells were transfected with Flag-mPer2 and with or without HA-mCry2-T4 or co-transfected with Flag-mPer2 and HA-mCry2-T5. Lysates were then immunoprecipitated (IP) with anti-Flag antibodies. Immunoprecipitates were separated by SDS- PAGE and then immunoblotted with antibodies against FLAG and HA as indicated. Left Panel: Input fractions. Right Panel: B and FT denote bound and flow through fractions, respectively. (D) HEK 293T cells were transfected with pBIND-mBmal1 and pACT-mCry2-T1/T2/T3/T4/T5 along with the pGL5luc reporter plasmid. Reporter activity was examined 24 h after transfection. Relative activities were calculated based on mock samples and values were plotted. Error bars indicate SEM from at least 3 experiments. * p < 0.01, ** p < 0.005, *** p < 0.0001 as determined by student's t-test compared to the control.

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