Extended Data Fig. 1: Development and optimization of Cal-ID in HEK293T cells.
From: Molecular recording of calcium signals via calcium-dependent proximity labeling
(a) A schematic diagram of the Cal-ID design used to screen BioID split sites (this early version used split BioID rather than TurboID). CaM: calmodulin, RS20: CaM-binding peptide. (b) Cal-ID activation results in HEK293T cells with 1 µM thapsigargin. Representative blot, n = 3 (independent). Biotin: 100 µM, 30 min incubation (Cal-ID), 1 hour incubation (BioID). Cal-ID constructs were transfected transiently. *: endogenous biotin carrier proteins, #: self-biotinylation of Cal-ID or BioID. Numbers: molecular weight (kDa). (c) Representative blots testing Cal-ID (T195/G196 split) with a self-inhibitory AviTag* motif in HEK293T cells with 3 µM thapsigargin and prolonged incubation. Representative blot, n = 3 (independent). Biotin: 100 µM, 1 hour incubation, transient transfection. (d) Representative blots comparing Cal-ID split site variants T195/G196 and L73/G74 for short Incubation. Both variants contain a self-inhibitory motif. Representative blot, n = 3 (independent). Transient transfection in HEK293T cells, 3 µM thapsigargin, biotin: 50 µM, 15 min incubation.
