gst keima cb5  (Addgene inc)

Journal: bioRxiv

Article Title: A multi-subunit autophagic capture complex facilitates degradation of ER stalled MHC-I in pancreatic cancer

doi: 10.1101/2024.10.27.620516

Figure Lengend Snippet: A. Western blot showing levels of the indicated proteins in non-PDAC (HEK293T and Hela) and PDAC (KP4, PaTu8902, Panc10.05, PaTu8988T) cells. B. Immunoblot for total levels of FLAG and Tapasin (input) and following immunoprecipitation (IP) of stably expressed HLA-A-FLAG in the indicated cell lines. Cells were pre-treated with 400 nM Bafilomycin A1 (BafA1) for 6 h to block degradation. C. Time course measurement of amino acid starvation (EBSS) induced ER-phagy flux in the indicated cell lines stably expressing Keima-cb5 relative to DMEM growth media. N = 6 independent experiments. P values were determined using a two-way analysis of variance. D. Flow cytometry-based quantification of plasma membrane levels of MHC-I (HLA-A, - B, -C) in KP4 cells upon amino acid starvation in EBSS for 9 or 22 h compared to DMEM. N = 3 independent experiments. Data are the mean ± S.D. P values were determined using a one-way analysis of variance. E. Immunoblot for the indicated total protein (input) and following immunoprecipitation (IP) of stably expressed HLA-A-FLAG in KP4 cells under full nutrient conditions (DMEM), treatment with 400 nM BafA1 for 6 h, amino acid (AA) starvation for 6 h (EBSS), or combined AA starvation and 400nM BafA1 treatment. Blots are representative of N = 3 independent experiments. F. Flow cytometry-based quantification of plasma membrane levels of MHC-I (HLA-A, -B, -C) after siRNA-mediated knockdown of TEX264, FAM134B, and CCPG1 in KP4 cells. N = 3 independent experiment. Data are the mean ± S.D. P values were determined using a one-way analysis of variance. G. Immunoblot of the indicated proteins in KP4 cells following siRNA-mediated knockdown of TEX264, FAM134B, and CCPG1. Blots are representative of N = 3 independent experiments. Quantification is relative to siCTRL. H. Immunofluorescence staining of KP4 cells for TEX264 (green), MHC-I (red), and DAPI (blue). Inset shows the magnified view of the region indicated by the white box. Scale bar: 10 µm.

Article Snippet: pLJM1-HLA-A-FLAG was generated by subcloning the pcDNA of HLA-A (Addgene #85162) together with 2× FLAG into the EcoRI and NheI sites of the pLJM1 lentiviral vector. pMXs-GFP-NBR1, pMXs-GFP-NBR1 ΔUBA, and pMXs-GFP-NBR1 ΔLIR were provided by J. Debnath. pMRX-INU-TEX264-FLAG and pMRX-INU-TEX264 LIR4A- FLAG were gifts from Noboru Mizushima (Addgene #128258 and #128259). pLJM1- Keima-cb5 was generated by cloning the Keima-cb5 fragment from pRK5-GST-Keima- cb5 (a gift from Carol Mercer; Addgene plasmid #137755) into the AgeI and EcoRI sites of the pLJM1 lentiviral vector. pHAGE-CMV-ENV-SAV10-GFP and pHAGE-CMV-ENV- GFP were developed by Peter M. Bruno.

Techniques: Western Blot, Immunoprecipitation, Stable Transfection, Blocking Assay, Expressing, Flow Cytometry, Membrane, Knockdown, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: A multi-subunit autophagic capture complex facilitates degradation of ER stalled MHC-I in pancreatic cancer

doi: 10.1101/2024.10.27.620516

Figure Lengend Snippet: A. Flow cytometry gating strategy for Keima-cb5 experiments shown in . B,C . Flow cytometry-based quantification of plasma membrane levels of MHC-I (HLA-A, -B, -C) in PaTu8988T ( B ) or Panc1 ( C ) cells following siRNA-mediated knockdown of TEX264, FAM138B, or CCPG1. N = 3 independent experiments. Data are the mean ± S.D. P values were determined using a one-way analysis of variance.

Article Snippet: pLJM1-HLA-A-FLAG was generated by subcloning the pcDNA of HLA-A (Addgene #85162) together with 2× FLAG into the EcoRI and NheI sites of the pLJM1 lentiviral vector. pMXs-GFP-NBR1, pMXs-GFP-NBR1 ΔUBA, and pMXs-GFP-NBR1 ΔLIR were provided by J. Debnath. pMRX-INU-TEX264-FLAG and pMRX-INU-TEX264 LIR4A- FLAG were gifts from Noboru Mizushima (Addgene #128258 and #128259). pLJM1- Keima-cb5 was generated by cloning the Keima-cb5 fragment from pRK5-GST-Keima- cb5 (a gift from Carol Mercer; Addgene plasmid #137755) into the AgeI and EcoRI sites of the pLJM1 lentiviral vector. pHAGE-CMV-ENV-SAV10-GFP and pHAGE-CMV-ENV- GFP were developed by Peter M. Bruno.

Techniques: Flow Cytometry, Membrane, Knockdown

Journal: bioRxiv

Article Title: A multi-subunit autophagic capture complex facilitates degradation of ER stalled MHC-I in pancreatic cancer

doi: 10.1101/2024.10.27.620516

Figure Lengend Snippet: A. Western blot showing levels of the indicated proteins in non-PDAC (HEK293T and Hela) and PDAC (KP4, PaTu8902, Panc10.05, PaTu8988T) cells. B. Immunoblot for total levels of FLAG and Tapasin (input) and following immunoprecipitation (IP) of stably expressed HLA-A-FLAG in the indicated cell lines. Cells were pre-treated with 400 nM Bafilomycin A1 (BafA1) for 6 h to block degradation. C. Time course measurement of amino acid starvation (EBSS) induced ER-phagy flux in the indicated cell lines stably expressing Keima-cb5 relative to DMEM growth media. N = 6 independent experiments. P values were determined using a two-way analysis of variance. D. Flow cytometry-based quantification of plasma membrane levels of MHC-I (HLA-A, - B, -C) in KP4 cells upon amino acid starvation in EBSS for 9 or 22 h compared to DMEM. N = 3 independent experiments. Data are the mean ± S.D. P values were determined using a one-way analysis of variance. E. Immunoblot for the indicated total protein (input) and following immunoprecipitation (IP) of stably expressed HLA-A-FLAG in KP4 cells under full nutrient conditions (DMEM), treatment with 400 nM BafA1 for 6 h, amino acid (AA) starvation for 6 h (EBSS), or combined AA starvation and 400nM BafA1 treatment. Blots are representative of N = 3 independent experiments. F. Flow cytometry-based quantification of plasma membrane levels of MHC-I (HLA-A, -B, -C) after siRNA-mediated knockdown of TEX264, FAM134B, and CCPG1 in KP4 cells. N = 3 independent experiment. Data are the mean ± S.D. P values were determined using a one-way analysis of variance. G. Immunoblot of the indicated proteins in KP4 cells following siRNA-mediated knockdown of TEX264, FAM134B, and CCPG1. Blots are representative of N = 3 independent experiments. Quantification is relative to siCTRL. H. Immunofluorescence staining of KP4 cells for TEX264 (green), MHC-I (red), and DAPI (blue). Inset shows the magnified view of the region indicated by the white box. Scale bar: 10 µm.

Article Snippet: pLJM1-HLA-A-FLAG was generated by subcloning the pcDNA of HLA-A (Addgene #85162) together with 2× FLAG into the EcoRI and NheI sites of the pLJM1 lentiviral vector. pMXs-GFP-NBR1, pMXs-GFP-NBR1 ΔUBA, and pMXs-GFP-NBR1 ΔLIR were provided by J. Debnath. pMRX-INU-TEX264-FLAG and pMRX-INU-TEX264 LIR4A- FLAG were gifts from Noboru Mizushima (Addgene #128258 and #128259). pLJM1- Keima-cb5 was generated by cloning the Keima-cb5 fragment from pRK5-GST-Keima- cb5 (a gift from Carol Mercer; Addgene plasmid #137755) into the AgeI and EcoRI sites of the pLJM1 lentiviral vector. pHAGE-CMV-ENV-SAV10-GFP and pHAGE-CMV-ENV- GFP were developed by Peter M. Bruno.

Techniques: Western Blot, Immunoprecipitation, Stable Transfection, Blocking Assay, Expressing, Flow Cytometry, Membrane, Knockdown, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: A multi-subunit autophagic capture complex facilitates degradation of ER stalled MHC-I in pancreatic cancer

doi: 10.1101/2024.10.27.620516

Figure Lengend Snippet: A. Flow cytometry gating strategy for Keima-cb5 experiments shown in . B,C . Flow cytometry-based quantification of plasma membrane levels of MHC-I (HLA-A, -B, -C) in PaTu8988T ( B ) or Panc1 ( C ) cells following siRNA-mediated knockdown of TEX264, FAM138B, or CCPG1. N = 3 independent experiments. Data are the mean ± S.D. P values were determined using a one-way analysis of variance.

Article Snippet: pLJM1-HLA-A-FLAG was generated by subcloning the pcDNA of HLA-A (Addgene #85162) together with 2× FLAG into the EcoRI and NheI sites of the pLJM1 lentiviral vector. pMXs-GFP-NBR1, pMXs-GFP-NBR1 ΔUBA, and pMXs-GFP-NBR1 ΔLIR were provided by J. Debnath. pMRX-INU-TEX264-FLAG and pMRX-INU-TEX264 LIR4A- FLAG were gifts from Noboru Mizushima (Addgene #128258 and #128259). pLJM1- Keima-cb5 was generated by cloning the Keima-cb5 fragment from pRK5-GST-Keima- cb5 (a gift from Carol Mercer; Addgene plasmid #137755) into the AgeI and EcoRI sites of the pLJM1 lentiviral vector. pHAGE-CMV-ENV-SAV10-GFP and pHAGE-CMV-ENV- GFP were developed by Peter M. Bruno.

Techniques: Flow Cytometry, Membrane, Knockdown

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: Control, Affinity Purification, Western Blot, Labeling, Mass Spectrometry, Quantitative Proteomics, Clone Assay