Western blot of proteins expressed from the two type VI secretion system operons in ΔT6SSpro strains.

Top panel, diagram of the two operons encoding elements of Type VI Secretion in A. tumefaciens. T6SSpro labels the intergenic gap that is deleted in ΔT6SSpro strains. Middle panel, western blots using anti-Hcp and anti-TssB in each of the indicated strains. Protein sizes (kDa) are shown on the right. Bottom panel, Coomassie stained gel showing total protein from each strain. Western blots were performed as described in the methods with the following modifications. Lysates were prepared by pelleting cells via centrifugation and resuspending in 1X loading buffer. Next, the suspension was run through a 20G needle for lysis. 3 gels were loaded with identical concentrations of sample. BlueStain2 Protein ladder (P008-500) was loaded into the first well of each. One of the three gels was Coomassie stained at room temperature with gentle shaking for 10–15 minutes and imaged for total protein content. The other two gels were transferred to immobilon-FL transfer membranes, blocked with 0.5% milk, and transferred to a solution of TBS + 0.05% Tween 20 with 4 μL of 1:1000 dilution of either anti-TssB or anti-Hcp for 1 hour. Membranes were washed and transferred to TBS + 0.05% Tween 20 with 4 μL of 1:1000 dilution of anti-rabbit HRP goat IgG for 1 hour. (TIF)