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Rab12 phosphorylation is sensitive to inhibitors of PKC. RBL cells (A-D) , or BMMCs (E, F) or LAD-2 cells (G, H) , were either sensitized with IgE and subsequently left untreated or incubated with either GSK2578215A, Go6976, <t>GF109203X</t> or LY333531 , as indicated, followed by cell trigger with antigen (DNP-HSA) for 5 minutes (A, B) , or Ion/TPA for 30 minutes (C-F) , or substance P for 2 minutes (G, H) . Cell lysates were analyzed by immunoblotting using anti phosphoRab12 antibodies followed by re-probing with anti Rab12 and anti GAPDH antibodies. Representative blots are shown. Blots were quantified by ImageJ software. Data is presented as the ratio between phosphoRab12 to Rab12 and normalized to untreated cells. Results are the mean ± SEM derived from 3 independent experiments. ***p.v<0.001, ****p.v<0.0001, assessed by one-way ANOVA. ns, not significant.
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Rab12 phosphorylation is sensitive to inhibitors of PKC. RBL cells (A-D) , or BMMCs (E, F) or LAD-2 cells (G, H) , were either sensitized with IgE and subsequently left untreated or incubated with either GSK2578215A, Go6976, GF109203X or LY333531 , as indicated, followed by cell trigger with antigen (DNP-HSA) for 5 minutes (A, B) , or Ion/TPA for 30 minutes (C-F) , or substance P for 2 minutes (G, H) . Cell lysates were analyzed by immunoblotting using anti phosphoRab12 antibodies followed by re-probing with anti Rab12 and anti GAPDH antibodies. Representative blots are shown. Blots were quantified by ImageJ software. Data is presented as the ratio between phosphoRab12 to Rab12 and normalized to untreated cells. Results are the mean ± SEM derived from 3 independent experiments. ***p.v<0.001, ****p.v<0.0001, assessed by one-way ANOVA. ns, not significant.

Journal: Frontiers in Immunology

Article Title: Phosphorylation-driven effector switching of Rab7 and Rab12 by the leucine-rich repeat kinase 1 in mast cells

doi: 10.3389/fimmu.2025.1709196

Figure Lengend Snippet: Rab12 phosphorylation is sensitive to inhibitors of PKC. RBL cells (A-D) , or BMMCs (E, F) or LAD-2 cells (G, H) , were either sensitized with IgE and subsequently left untreated or incubated with either GSK2578215A, Go6976, GF109203X or LY333531 , as indicated, followed by cell trigger with antigen (DNP-HSA) for 5 minutes (A, B) , or Ion/TPA for 30 minutes (C-F) , or substance P for 2 minutes (G, H) . Cell lysates were analyzed by immunoblotting using anti phosphoRab12 antibodies followed by re-probing with anti Rab12 and anti GAPDH antibodies. Representative blots are shown. Blots were quantified by ImageJ software. Data is presented as the ratio between phosphoRab12 to Rab12 and normalized to untreated cells. Results are the mean ± SEM derived from 3 independent experiments. ***p.v<0.001, ****p.v<0.0001, assessed by one-way ANOVA. ns, not significant.

Article Snippet: GSK2578215A (cat #4629) and GF109203X (cat #0741) were from Tocris Bioscience (Bristol, UK).

Techniques: Phospho-proteomics, Incubation, Western Blot, Software, Derivative Assay