gst rfp cb5  (Addgene inc)

Journal: Autophagy

Article Title: Mass spectrometry proteomics reveals a function for mammalian CALCOCO1 in MTOR-regulated selective autophagy

doi: 10.1080/15548627.2020.1719746

Figure Lengend Snippet: CALCOCO1 has a role in reticulophagy. (A) GSTLSCSGFP-cb5 reticulophagy assay in HEK293 control and 2 sgCALCOCO1 KO cell lines, treated with Veh, 100 nM MLN, or glucose (Glc) starved. GST-tagged proteins/peptides in cell lysates were captured by glutathione agarose affinity purification. Ratio of the bottom fragment to the full-length fusion protein is shown, normalized to MLN in control cells. CALCOCO1 in cell lysates is shown. “B2” and “C3” represent different targets for guide RNA. *nonspecific fragment (B) GSTLSCSGFP-cb5 reticulophagy assay in HEK293 control and sgCALCOCO1 KO cells, treated with Veh, MLN, 10 ug/ml tunicamycin (Tun), or with galactose (Gal). Top panel. GST-tagged proteins/peptides in the LMF. Ratio of the bottom fragment to the full-length fusion protein is shown, normalized to MLN in control cells. Bottom panel. Immunoblot analyses of CALCOCO1 in LMF and high-speed supernatant (HSS) fractions. *nonspecific fragment (C) Immunoblot analysis of GST-Keima-cb5 assay in HEK293 cells, treated with Veh, MLN, and 5 ug/ml Tun as labeled. Ratio of 25 kD fragment to the full-length fusion protein is shown, normalized to MLN in control cells. (D) Relative protein abundances in sgCALCOCO1 KO HEK293 cells compared to sgGFP (GFP) control WT cells (mean, n = 5). Cells were treated for 24 h with 100 nM MLN0128. Proteins were quantified by label-free mass spectrometry. P-values for the protein abundance changes were determined by a two-sided Student’s t-test. (E) GST-BHMT autophagy assay in control and sgCALCOCO1 KO clones, with treatments as in Figure 6A. Ratio of BHMT fragment to GFP, normalized to MLN in control cells, is shown. Immunoblots were probed with antibodies as described. (F) Immunoblot analysis of WT MB231 and 3 sgCALCOCO1 KO cell lines, with antibodies, as shown. Cells were treated for 48 h with Veh, 100 nM MLN, and 20 nM bafilomycin A1, as shown

Article Snippet: GST - RFP-cb5 and GST-Keima-cb5 (Addgene, 137754 and 137755; deposited by Carol Mercer) were made by PCR of RFP or Keima from existing plasmids, using sequence-specific primers with recognition sites for NotI and XbaI restriction enzymes (RE).

Techniques: Affinity Purification, Western Blot, Labeling, Mass Spectrometry, Clone Assay