Figure 1: Ca²⁺ triggers recruitment of ESCRT III complex at the cell membrane.

SILAC labelled and unlabelled C2C12 myoblasts treated for 5 min with Ionomycin (10 μM) or with DMSO (0.1%) were pooled, and the cell membrane proteins were extracted. (a) A representative coomassie-stained 1D SDS-PAGE gel (n=6) used for differential cell membrane proteomic analysis. The gel shows molecular weight marker and the cell membrane protein pool (pooled lysate). The gel lane for pooled lysate was cut into slices and processed for proteomic analysis by mass spectrometry (MS). (b) Scatter (volcano) plot shows the fold change (log₂ average ratio) and –log₁₀ P value (one sample t-test versus ratio of 1.0) for the proteins quantified in at least two of the six independently replicated experiments. Black spots represent protein whose abundance was not altered in response to ionomycin-triggered Ca²⁺ increase or the alteration did not reach the designated statistical significance across replicates. Red symbols represent proteins with P<0.06 (one sample t-test versus ratio of 1.0) and fold change ±0.4 s.d., whereas blue symbols mark proteins with P<0.06 and fold change ±0.2 s.d. Green symbols show the ESCRT and related proteins that show fold change greater than ±0.2 s.d. (c) Western blot analysis of change in the cell membrane level of representative non-ESCRT proteins that were identified by MS analysis to be increased (Annexin A1- and Galectin-1) or not altered (Cadherin) at the cell surface by ionomycin-induced Ca²⁺ increase. The molecular weight (KDa) markers are marked for each blot. (d) Plot showing average change in cell surface level of ESCRT proteins as assessed by MS analysis. Specific protein level were quantified by ion chromatogram of peptides (generated by IP2 Census quantitation software) such that the values for all peptides corresponding to a protein were averaged to obtain ratio shown for the abundance of this protein in Ionomycin versus DMSO treated cells—error bars represent s.d. (e) Representative western blots (n=3) with corresponding molecular weight (KDa) markers, and (f) averaged ratio for ionomycin triggered change in cell surface level of proteins in ESCRT III and accessory proteins—error bars represent s.e.