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: Figure 5. CALCOCO1 interacts with MAP1LC3C and other LC3/GABARAP family members. (A) Cartoon of domains and motifs found in human and mouse CALCOCO family members. (B) Immunoprecipitation (IP) analyses of CALCOCO1-MYCDDK and HA-tagged MAP1LC3/GABARAP orthologs in HEK293 cells. HA-tagged protein complexes were immunoprecipitated with anti-HA antibody, resolved by SDS-PAGE, and probed with anti-HA or anti-DDK antibody. Expressions of tagged proteins and ACTB in cell lysates are shown with antibodies as described. (C) IP analyses of CALCOCO1-MYCDDK and HA-tagged MAP1LC3/GABARAP orthologs expressed in HEK293 cells that were treated overnight with chloroquine (CQ) to inhibit lysosomal flux. HA-tagged protein complexes were IP’d with anti-HA antibody and probed with anti-DDK or mixed antibodies against HA, MAP1LC3A/B, and MAP1LC3C. Expressions of tagged proteins and ACTB in cell lysates are shown with antibodies as described. (D) IP analyses of HA-MAP1LC3C or HA-GFP co-expressed with wild type (WT) CALCOCO1-MYCDDK, CLIR mutants L140A and V142A, and LIR mutant W47A in HEK293 cells. CALCOCO1W47A was transfected at 2 different concentrations to match WT expression levels. HA-tagged protein complexes were immunoprecipitated with anti-HA antibody, and probed with anti-HA or anti-CALCOCO1 antibody. Expressions of tagged proteins and ACTB in cell lysates are shown with antibodies as noted. (E) IP analyses of CALCOCO1-MYCDDK with endogenous MAP1LC3B. WT CALCOCO1-MYCDDK, 2 concentrations of the CALCOCO1W47A, and GFP-DDK (control) were expressed in HEK293 cells, then treated for 18 h with 100 nM MLN0128 and 100 uM CQ. CALCOCO1-MYCDDK complexes were immunoprecipitated with anti- Flag® M2 affinity gel (MilliporeSigma, A2220) and probed with anti-DDK or MAP1LC3B antibodies. Expressions of CALCOCO1-MYCDDK, GFP-DDK, ACTB and MAP1LC3B in cell lysates are shown with antibodies as noted. (F) IP analyses of HA-MAP1LC3C or HA-GFP co-expressed with WT CALCOCO1-MYCDDK, CLIR mutants (L140A and V142A), and R12H mutant in HEK293 cells. HA-tagged protein complexes were immunoprecipitated with anti-HA antibody and probed with anti-HA or anti- CALCOCO1 antibody. Expressions of tagged proteins and ACTB in cell lysates are shown with antibodies, as noted.
Article Snippet: Primary antibodies used in this study include: Abcam antibodies for CALCOCO1/CoCoA (ab70564); ABD SeroTech antibodies GAPDH (HCA272); BD Biosciences antibodies for SQSTM1 (610832) and RPS6KB1 (611261); Cell Signaling Technology antibodies for ATG3 (3415), ATG5 (8540), ATG7 (8558), MAP1LC3A/B (12741), MAP1LC3C (14723), ULK1 (6439 and 8054), p-EIF4EBP1 T37/46 (2855), p-EIF4EBP1 S65 (9456), EIF4EBP1 (9644) p-AKT1 S473 (4060), AKT1 (9272) p-RPS6KB T389 (9234), p-ACACA S79 (11818), ACACA (3662) DYKDDDDK-tag (14793), MYC-tag (2276), ACTB (beta-Actin; 3700), LMNA (lamin A/C; 4777); Novus antibodies for MAP1LC3B (NB100-2220); Protein Tech antibodies for RETREG1 (21537-1-AP), GABARAPL2 (18724-1-AP), MAP1LC3C (18726-1-AP); Santa Cruz Biotechnology antibodies for CALCOCO1 (sc-515670), HAtag (sc-805), GST-tag (sc-138), TUBA (alpha-tubulin; sc5286); MilliporeSigma antibody for Flag M2 (F1804).
Techniques: Immunoprecipitation, SDS Page, Mutagenesis, Transfection, Expressing, Control
Thermo Fisher is a verified supplier 
Thermo Fisher manufactures this product 
