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. 2012 May 16;287(29):24164–24173. doi: 10.1074/jbc.M112.341644

FIGURE 4.

FIGURE 4.

Analysis of tic22 and alr0115 expression in the mutant. A, RNA from wild-type Anabaena sp. PCC 7120 was isolated and either rnpB or the indicated intergenic regions amplified by RT-PCR in the presence (+) or absence of reverse transcriptase (−). Primers used are indicted in Fig. 3A. B, the transcript abundance of the downstream genes was analyzed in AFS-I-tic22 (AFS-I) by RT-PCR and compared with the transcript abundance seen in WT. The combinations of oligonucleotides are indicated, and rnpB (lanes 9 and 10) was amplified as control. C, indicated strains were spotted on BG11 plates (left) with 30 mm lysozyme as described in B. For immunoblot (bottom), similar amounts of cells were subjected to SDS-PAGE and immunodecorated with anaTic22 antibodies. D, identical amounts of cell lysate from indicated strains were subjected to immunodecoration by anaTic22 and AtpB antibodies. E, same samples as in D were subjected to immunodecoration by anaOmp85 antibodies. The cell extract from Anabaena sp. was incubated without (left) or with anaOmp85-POTRA-His (right), immobilized on NTA. Eluted protein was subjected to SDS-PAGE followed by Coomassie Blue staining. F, expressed anaOmp85-POTRA-His (lanes 1–3) and anaTic22-His (lanes 3–5) incubated with glutaraldehyde (lanes 2–4) was subjected to SDS-PAGE followed by Coomassie Blue staining. anaOmp85-POTRA (black), anaTic22 (white), and POTRA-anaTic22 (gray) cross-links are highlighted by triangles. G, Anabaena cells were incubated with formaldehyde, solubilized (I, lanes 1 and 6), and incubated with antibodies against anaOmp85 (lanes 7–10). After affinity purification, the flow-through (F, lanes 2 and 7), wash (W, lanes 3, 4, 8, and 9), and elution fraction (E, lanes 5 and 10) was collected and subjected to SDS-PAGE followed by Coomassie Blue staining. The region of lane 10 indicated (arrowheads) were excised, boiled, and subjected to SDS-PAGE, blotted, and immunodecorated with anaTic22 antibodies (lanes 11 and 12). The migration of the heavy chain (HC) and light chain (LC) of the antibody (black arrowhead) and of anaTic22 (white arrowhead) is indicated.