Journal: The Journal of Biological Chemistry

Article Title: Cyanophycinase is required for heterotrophy in cyanobacteria

doi: 10.1016/j.jbc.2025.110791

Figure Lengend Snippet: ArgD binds CphB in both LAH and PAT conditions. A , the relative levels of ArgD and CphB under PAT and LAH in WT and Δ cphB were determined by immunodetection. Whole-cell lysates were separated on SDS-PAGE and blotted onto a PVDF membrane, and the ArgD and CphB proteins were detected using specific antibodies. The Ponceau staining of the membrane is shown for the loading control. Repetitions of the experiment and their statistical analysis are presented in . B , coimmunopurification of f.ArgD with CphB was performed using the same amounts of PAT- or LAH-grown f.argD/ Δ argD cells. The eluates were separated by SDS-PAGE together with the input lysates, including 50% of the lysate from the PAT-grown cells (PAT 50 ). The SYPRO-stained gel was subsequently blotted to a PVDF membrane, which was probed with specific antibodies against CphB and the FLAG tag of ArgD. The control f.ArgD pull-down prepared from the Δ cphB cells is shown in Ref. . CphB, cyanophycinase; f.ArgD, FLAG-tagged variant of ArgD; LAH, light-activated heterotrophic; PAT, photoautotroph; PVDF, polyvinylidene fluoride.

Article Snippet: The proteins were separated, visualized with SYPRO orange protein dye (Lumiprobe ProteOrange, catalog no.: 40210) and transferred onto a polyvinylidene fluoride membrane (Sigma–Aldrich, Immobilon-P, catalog no.: IPVH00010) that was subsequently incubated with primary anti-CphB and anti-ArgD antibodies ( ).

Techniques: Immunodetection, SDS Page, Membrane, Staining, Control, FLAG-tag, Variant Assay