Figure 1: Functional interaction of Beclin1 with ROCK1 upon metabolic stress. | Nature Communications

Figure 1: Functional interaction of Beclin1 with ROCK1 upon metabolic stress.

From: Identification of ROCK1 kinase as a critical regulator of Beclin1-mediated autophagy during metabolic stress

Figure 1

(a) Identification of Beclin1 as a substrate of ROCK1 via affinity purification. Left panel: HeLa cells growing in control (C: nutrient-rich media) or starved in HBSS (H) for 4 h were lysed and subjected to IP with ROCK1 cross-linked beads. Proteins bound were eluted and detected by SDS–PAGE, followed by silver staining that presents precipitated proteins identified by LC-MS/MS. Right panel: HeLa cells grown in control media or HBSS (4 h) were collected, whole cell lysates prepared and immunoprecipitated with ROCK1 cross-linked beads. The resulting immune complexes were divided into two, and were subjected to SDS–PAGE and analysed for Beclin1 (MES buffer) and ROCK1 (MOPS buffer). Inputs and IP were analysed by western blotting. HBSS-treated cell extracts were used for IgG IP controls. Whole cell lysates (10%) were run for inputs. Total cell lysates were rerun and analysed for LC3 and β-actin. (b) Co-IP was performed with 293T whole cell extracts by using ROCK1-crosslinked agarose, and the resulting IP was blotted with indicated antibodies. 293T cells transfected with Flag-Beclin1 for 36 h, cultured in control media or starved (4 h), were immunoprecipitated with anti-Flag beads. Inputs and immune complexes were resolved and detected. Total cell lysates were rerun and examined for LC3 and β-actin expression. (c) Interaction of YFP-ROCK1 and CFP-Beclin1 was examined by using live cell spinning disk microscopy and FRET in live cells. Graph represents FRETAverage ratio changes (YFP-ROCK1/CFP-Beclin1) upon starvation. Data represents mean±s.e.m.; n=6. Three different experiments were performed. For the control, the right top graph represents CFP intensity over time (corrected for photobleach), and the right bottom right represents YFP intensity (corrected for photobleach). In both control FRET experiments, no significant change was observed in fluorescence, in the presence or absence of HBSS medium. P values were calculated using Student's t-test.

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