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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Mol Microbiol. 2014 Jul 16;93(4):682–697. doi: 10.1111/mmi.12681

Figure 2. Each of CarD’s functional domains is required for optimal growth in mycobacteria.

Figure 2

A. Immunoprecipitation experiments with a monoclonal antibody specific for HA in the M. smegmatis strains expressing CarDWT-HA (lane 1), CarDR25E-HA (lane 2), CarDK90A-HA (lane 3), CarDK90E-HA (lane 4), CarDW85A-HA (lane 5), CarDK125A-HA (lane 6), or CarDK125E-HA (lane 7). Inputs (before immunoprecipitation) and eluates were analyzed by western blotting with antibodies specific for either RNAP β (Panel A and D) or CarD (Panel B, C, and E). Panel C is a longer exposure of the film from panel B in order to show the CarDK90E band.

B–C. The doubling time of each CarD strain was expressed as a ratio to the doubling time of the CarDWT expressing strain for the M. smegmatis strains expressing CarDWT, CarDR25E, CarDK90A, CarDW85A, CarDK125A, or CarDK125E (B) and M. tuberculosis strains expressing CarDWT, CarDK125A, or CarDR47E (C). Each graph shows the mean ± SEM of data from at least three replicates. Significance of the differences between mutant strains and WT were determined by calculating P values by Student’s t test. An asterisk indicates significance with a P value of <0.05, two asterisks indicate significance with a P value of <0.01, and three asterisks indicate significance with a P value of <0.005.

D. Plated dilutions of M. smegmatis strains expressing CarDWT, CarDR25E, CarDW85A, CarDK90A, CarDK125A, or CarDK125E on LB after 3 days of growth at 37°C.

E. Image of nondenaturing polyacrylamide gel from EMSAs with no protein, M. tuberculosis CarDWT, CarDR25E, or CarDR47E incubated with IRDye labeled M. smegmatis rrnAPL DNA. The reactions were separated on a nondenaturing polyacrylamide gel, which was then imaged using the Odyssey CLX imaging system (LI-COR).