Figure 2
From: Proteomics-Based Approach Identifies Altered ER Domain Properties by ALS-Linked VAPB Mutation
Proteomic analysis of the immunoprecipitates for mRFP-Climp63 and mRFP-DP1 isolated from N2a cells in a detergent-free condition. (A) Schematic procedure of immuno-isolation and proteomics of different ER domains. The cell lines were homogenized in detergent-free sucrose buffer and subjected to immunoprecipitation (IP), followed by Western blotting or LC-MS. (B) Western blotting of the immunoprecipitates by anti-mRFP antibodies (3G8 and 8D6) from N2a cell lines expressing mRFP, mRFP-Climp63 and mRFP -DP1. Precipitated proteins were analyzed by antibodies for indicated proteins (*non-specific bands). (C-E) Obtained immunoprecipitates were digested with trypsin and analyzed by LC-MS. Proteome Discoverer version 2.2 was used to identify the proteins and quantify their abundancies (label free quantification). After normalization with the amount of precipitated mRFP-tagged proteins, protein abundancies were compared between the two IP sets and log2 values of the fold changes (FCs; mRFP-Climp63-IP/mRFP-DP1-IP) were calculated. (C) Scatter plot of log2FCs for two IP sets (3G5 and 8D6). Proteins enriched by mRFP-Climp63-IP (red) or mRFP-DP1-IP (green) in two biological replicates (D) were colorized and known sheets and tubule proteins are indicated. (D) Identified proteins enriched in mRFP-Climp63-IP (upper diagram) or in mRFP-DP1-IP (lower diagram) in two biological replicates (rep 1 and 2); total 194 and 50 proteins were identified in both replicates, respectively. (E) Identified proteins were processed for functional annotation analysis. Proteins related to nucleus were relatively abundant in the mRFP-Climp63-IP, whereas proteins related to ER lumen, Golgi apparatus and chaperone were abundant in the mRFP-DP1-IP.
