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plko pgk rtta p2a cre sbe nls mscarlet  (Addgene inc)
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Addgene inc plko pgk rtta p2a cre sbe nls mscarlet
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Plko Pgk Rtta P2a Cre Sbe Nls Mscarlet, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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paper addgene 200580  (Addgene inc)
91
Addgene inc paper addgene 200580
Figure 1. The LIM-peptidase region of <t>DAR4</t> functions as an integrated decoy (A) Diagram of known protein motifs of DAR4 and mutant and chimeric forms used to test hypersensitive responses. TIR, Toll, and interleukin-1 receptor ho- mology regions; NB, NB-ARC nucleotide-binding; ARC, APAF-1,R proteins, and CED-4 domains; LRR, leucine-rich-repeat domain; LIM, LIN11, Isl1, and MEC3 domains; each Zn finger of the two LIM domains is represented; PEP, zinc-metallopeptidase region with AHEMMHA-EE active site; GST, glutathione S-transferase; GFP, green fluorescent protein. Red letters indicate the positions of mutated amino acid codons. C1340Y is in the fourth Zn-finger of the LIM repeats. H1488, H1492, E1508, and E1509 are in the HEMMA and EE motifs of the peptidase active site. The diagram is not to scale. Figure S2 shows the amino acid sequences and motifs of the <t>DA1</t> family. (B and C) Hypersensitive responses in N. tabacum leaves were assessed at 4 days post-infection. Images represent typical samples of three replicates. DAR4 and variants in (A) were co-expressed as <t>3HA-tagged</t> proteins with (B) or without (C) CSA1. Quantified HR responses29 are shown by the stacked bars are color coded to show the percentage of each cell death scale (0–5) in triplicated samples. Yellow represents the presence of HR, and green represents the absence of HR. (D) Immunoblots showing expression levels of 3HA-tagged CSA1 and CHS3/DAR4 variants in leaf infection assays in the left panel and dar4-LPGFP in the right immunoblot. 3HA-DA1 proteins were detected by HRP-coupled anti-HA antibodies, and GFP-tagged dar4-LPGFP was detected with HRP-coupled anti-GFP antibodies. To accommodate large differences in expression of 3HA-DAR4 deletions and mutants, different loadings on separate gels were electrophoresed in order to detect protein bands.
Paper Addgene 200580, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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paper addgene 200560 paav tbg ces2c dc s230a plasmid  (Addgene inc)
93
Addgene inc paper addgene 200560 paav tbg ces2c dc s230a plasmid
Figure 1. The LIM-peptidase region of <t>DAR4</t> functions as an integrated decoy (A) Diagram of known protein motifs of DAR4 and mutant and chimeric forms used to test hypersensitive responses. TIR, Toll, and interleukin-1 receptor ho- mology regions; NB, NB-ARC nucleotide-binding; ARC, APAF-1,R proteins, and CED-4 domains; LRR, leucine-rich-repeat domain; LIM, LIN11, Isl1, and MEC3 domains; each Zn finger of the two LIM domains is represented; PEP, zinc-metallopeptidase region with AHEMMHA-EE active site; GST, glutathione S-transferase; GFP, green fluorescent protein. Red letters indicate the positions of mutated amino acid codons. C1340Y is in the fourth Zn-finger of the LIM repeats. H1488, H1492, E1508, and E1509 are in the HEMMA and EE motifs of the peptidase active site. The diagram is not to scale. Figure S2 shows the amino acid sequences and motifs of the <t>DA1</t> family. (B and C) Hypersensitive responses in N. tabacum leaves were assessed at 4 days post-infection. Images represent typical samples of three replicates. DAR4 and variants in (A) were co-expressed as <t>3HA-tagged</t> proteins with (B) or without (C) CSA1. Quantified HR responses29 are shown by the stacked bars are color coded to show the percentage of each cell death scale (0–5) in triplicated samples. Yellow represents the presence of HR, and green represents the absence of HR. (D) Immunoblots showing expression levels of 3HA-tagged CSA1 and CHS3/DAR4 variants in leaf infection assays in the left panel and dar4-LPGFP in the right immunoblot. 3HA-DA1 proteins were detected by HRP-coupled anti-HA antibodies, and GFP-tagged dar4-LPGFP was detected with HRP-coupled anti-GFP antibodies. To accommodate large differences in expression of 3HA-DAR4 deletions and mutants, different loadings on separate gels were electrophoresed in order to detect protein bands.
Paper Addgene 200560 Paav Tbg Ces2c Dc S230a Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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paper n a recombinant dna gecko v2 crispr grna library sanjana  (Addgene inc)
93
Addgene inc paper n a recombinant dna gecko v2 crispr grna library sanjana
Figure 1. The LIM-peptidase region of <t>DAR4</t> functions as an integrated decoy (A) Diagram of known protein motifs of DAR4 and mutant and chimeric forms used to test hypersensitive responses. TIR, Toll, and interleukin-1 receptor ho- mology regions; NB, NB-ARC nucleotide-binding; ARC, APAF-1,R proteins, and CED-4 domains; LRR, leucine-rich-repeat domain; LIM, LIN11, Isl1, and MEC3 domains; each Zn finger of the two LIM domains is represented; PEP, zinc-metallopeptidase region with AHEMMHA-EE active site; GST, glutathione S-transferase; GFP, green fluorescent protein. Red letters indicate the positions of mutated amino acid codons. C1340Y is in the fourth Zn-finger of the LIM repeats. H1488, H1492, E1508, and E1509 are in the HEMMA and EE motifs of the peptidase active site. The diagram is not to scale. Figure S2 shows the amino acid sequences and motifs of the <t>DA1</t> family. (B and C) Hypersensitive responses in N. tabacum leaves were assessed at 4 days post-infection. Images represent typical samples of three replicates. DAR4 and variants in (A) were co-expressed as <t>3HA-tagged</t> proteins with (B) or without (C) CSA1. Quantified HR responses29 are shown by the stacked bars are color coded to show the percentage of each cell death scale (0–5) in triplicated samples. Yellow represents the presence of HR, and green represents the absence of HR. (D) Immunoblots showing expression levels of 3HA-tagged CSA1 and CHS3/DAR4 variants in leaf infection assays in the left panel and dar4-LPGFP in the right immunoblot. 3HA-DA1 proteins were detected by HRP-coupled anti-HA antibodies, and GFP-tagged dar4-LPGFP was detected with HRP-coupled anti-GFP antibodies. To accommodate large differences in expression of 3HA-DAR4 deletions and mutants, different loadings on separate gels were electrophoresed in order to detect protein bands.
Paper N A Recombinant Dna Gecko V2 Crispr Grna Library Sanjana, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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addgene plasmid  (Addgene inc)
93
Addgene inc addgene plasmid
Figure 1. The LIM-peptidase region of <t>DAR4</t> functions as an integrated decoy (A) Diagram of known protein motifs of DAR4 and mutant and chimeric forms used to test hypersensitive responses. TIR, Toll, and interleukin-1 receptor ho- mology regions; NB, NB-ARC nucleotide-binding; ARC, APAF-1,R proteins, and CED-4 domains; LRR, leucine-rich-repeat domain; LIM, LIN11, Isl1, and MEC3 domains; each Zn finger of the two LIM domains is represented; PEP, zinc-metallopeptidase region with AHEMMHA-EE active site; GST, glutathione S-transferase; GFP, green fluorescent protein. Red letters indicate the positions of mutated amino acid codons. C1340Y is in the fourth Zn-finger of the LIM repeats. H1488, H1492, E1508, and E1509 are in the HEMMA and EE motifs of the peptidase active site. The diagram is not to scale. Figure S2 shows the amino acid sequences and motifs of the <t>DA1</t> family. (B and C) Hypersensitive responses in N. tabacum leaves were assessed at 4 days post-infection. Images represent typical samples of three replicates. DAR4 and variants in (A) were co-expressed as <t>3HA-tagged</t> proteins with (B) or without (C) CSA1. Quantified HR responses29 are shown by the stacked bars are color coded to show the percentage of each cell death scale (0–5) in triplicated samples. Yellow represents the presence of HR, and green represents the absence of HR. (D) Immunoblots showing expression levels of 3HA-tagged CSA1 and CHS3/DAR4 variants in leaf infection assays in the left panel and dar4-LPGFP in the right immunoblot. 3HA-DA1 proteins were detected by HRP-coupled anti-HA antibodies, and GFP-tagged dar4-LPGFP was detected with HRP-coupled anti-GFP antibodies. To accommodate large differences in expression of 3HA-DAR4 deletions and mutants, different loadings on separate gels were electrophoresed in order to detect protein bands.
Addgene Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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paper n a recombinant dna dcas9 krab sgrna vector zhang  (Addgene inc)
94
Addgene inc paper n a recombinant dna dcas9 krab sgrna vector zhang
Figure 1. The LIM-peptidase region of <t>DAR4</t> functions as an integrated decoy (A) Diagram of known protein motifs of DAR4 and mutant and chimeric forms used to test hypersensitive responses. TIR, Toll, and interleukin-1 receptor ho- mology regions; NB, NB-ARC nucleotide-binding; ARC, APAF-1,R proteins, and CED-4 domains; LRR, leucine-rich-repeat domain; LIM, LIN11, Isl1, and MEC3 domains; each Zn finger of the two LIM domains is represented; PEP, zinc-metallopeptidase region with AHEMMHA-EE active site; GST, glutathione S-transferase; GFP, green fluorescent protein. Red letters indicate the positions of mutated amino acid codons. C1340Y is in the fourth Zn-finger of the LIM repeats. H1488, H1492, E1508, and E1509 are in the HEMMA and EE motifs of the peptidase active site. The diagram is not to scale. Figure S2 shows the amino acid sequences and motifs of the <t>DA1</t> family. (B and C) Hypersensitive responses in N. tabacum leaves were assessed at 4 days post-infection. Images represent typical samples of three replicates. DAR4 and variants in (A) were co-expressed as <t>3HA-tagged</t> proteins with (B) or without (C) CSA1. Quantified HR responses29 are shown by the stacked bars are color coded to show the percentage of each cell death scale (0–5) in triplicated samples. Yellow represents the presence of HR, and green represents the absence of HR. (D) Immunoblots showing expression levels of 3HA-tagged CSA1 and CHS3/DAR4 variants in leaf infection assays in the left panel and dar4-LPGFP in the right immunoblot. 3HA-DA1 proteins were detected by HRP-coupled anti-HA antibodies, and GFP-tagged dar4-LPGFP was detected with HRP-coupled anti-GFP antibodies. To accommodate large differences in expression of 3HA-DAR4 deletions and mutants, different loadings on separate gels were electrophoresed in order to detect protein bands.
Paper N A Recombinant Dna Dcas9 Krab Sgrna Vector Zhang, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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paper rrid addgene 203845 plenti hubc cre gfp gift  (Addgene inc)
91
Addgene inc paper rrid addgene 203845 plenti hubc cre gfp gift
Figure 1. The LIM-peptidase region of <t>DAR4</t> functions as an integrated decoy (A) Diagram of known protein motifs of DAR4 and mutant and chimeric forms used to test hypersensitive responses. TIR, Toll, and interleukin-1 receptor ho- mology regions; NB, NB-ARC nucleotide-binding; ARC, APAF-1,R proteins, and CED-4 domains; LRR, leucine-rich-repeat domain; LIM, LIN11, Isl1, and MEC3 domains; each Zn finger of the two LIM domains is represented; PEP, zinc-metallopeptidase region with AHEMMHA-EE active site; GST, glutathione S-transferase; GFP, green fluorescent protein. Red letters indicate the positions of mutated amino acid codons. C1340Y is in the fourth Zn-finger of the LIM repeats. H1488, H1492, E1508, and E1509 are in the HEMMA and EE motifs of the peptidase active site. The diagram is not to scale. Figure S2 shows the amino acid sequences and motifs of the <t>DA1</t> family. (B and C) Hypersensitive responses in N. tabacum leaves were assessed at 4 days post-infection. Images represent typical samples of three replicates. DAR4 and variants in (A) were co-expressed as <t>3HA-tagged</t> proteins with (B) or without (C) CSA1. Quantified HR responses29 are shown by the stacked bars are color coded to show the percentage of each cell death scale (0–5) in triplicated samples. Yellow represents the presence of HR, and green represents the absence of HR. (D) Immunoblots showing expression levels of 3HA-tagged CSA1 and CHS3/DAR4 variants in leaf infection assays in the left panel and dar4-LPGFP in the right immunoblot. 3HA-DA1 proteins were detected by HRP-coupled anti-HA antibodies, and GFP-tagged dar4-LPGFP was detected with HRP-coupled anti-GFP antibodies. To accommodate large differences in expression of 3HA-DAR4 deletions and mutants, different loadings on separate gels were electrophoresed in order to detect protein bands.
Paper Rrid Addgene 203845 Plenti Hubc Cre Gfp Gift, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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id nk698  (Addgene inc)
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Addgene inc id nk698

Id Nk698, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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p1303 dv sftpc tdtomato  (Addgene inc)
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paper n a pegfp ccpg1 dntd human ccpg1  (Addgene inc)
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Figure 1. <t>CCPG1</t> Is an LIR Motif-Containing Interactor of Human ATG8 Orthologs (A) Schematic of CCPG1 structure (NTD, N-terminal amino acids 1–230; TM, transmembrane anchor). (B) GST or GST fusions of ATG8 orthologs (LC3B, LC3C, and GABARAP) were used in affinity precipitation (AP) of transfected myc-CCPG1 from HEK293 cells. (C) GST or GST-GABARAP (mtLDS, LIR-docking site mutant) were used in AP of transfected myc-CCPG1 NTD from HEK293 cells.
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paper n a primer  (Novus Biologicals)
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Novus Biologicals paper n a primer
Figure 1. <t>CCPG1</t> Is an LIR Motif-Containing Interactor of Human ATG8 Orthologs (A) Schematic of CCPG1 structure (NTD, N-terminal amino acids 1–230; TM, transmembrane anchor). (B) GST or GST fusions of ATG8 orthologs (LC3B, LC3C, and GABARAP) were used in affinity precipitation (AP) of transfected myc-CCPG1 from HEK293 cells. (C) GST or GST-GABARAP (mtLDS, LIR-docking site mutant) were used in AP of transfected myc-CCPG1 NTD from HEK293 cells.
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Image Search Results


Key resources table

Journal: iScience

Article Title: A mechanistic basis for the malignant progression of salivary gland tumors

doi: 10.1016/j.isci.2021.103508

Figure Lengend Snippet: Key resources table

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Chicken polyclonal anti-GFP Abcam Ab13970; RRID: AB_300798 Rabbit monoclonal anti-E-cadherin (clone 24E10) Cell Signaling Technology 3195; RRID: AB_2291471 Goat polyclonal anti-HMGA2 R&D Systems AF3184 Goat polyclonal anti-SOX2 R&D Systems AF2018; RRID: AB_355110 Mouse monoclonal anti-Vimentin Developmental Studies Hybridoma Bank 40E-C; RRID: AB_528504 Rabbit polyclonal anti-Aquaporin 5 Millipore AB15858; RRID: AB_992731 Rat monoclonal anti-Keratin 19 Developmental Studies Hybridoma Bank TROMA-III; RRID: AB_2133570 Rabbit polyclonal anti-Active Caspase-3 R&D Systems AF835; RRID: AB_2243952 Rat monoclonal anti-integrin α6 (clone GoH3) BD Biosciences 555734; RRID: AB_2296273 Chemicals, peptides, and recombinant proteins 5-ethynyl-2’-deoxyuridine (EdU) ThermoFisher Scientific A10044 Tamoxifen Sigma T5648 Critical commercial assays Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye ThermoFisher Scientific C10337 Experimental models: Cell lines Lenti-X 293T cell line Takara/Clontech 632180 Experimental models: Organisms/strains Tg(tetO-HRAS)65Lc NCI Mouse Repository Stock No. 01XB4 Gt(ROSA)26Sor tm1(EYFP)Cos The Jackson Laboratory Stock No. 006148 Recombinant DNA pMD2.G Dr. Didier Trono Addgene #12259 psPAX2 Dr. Didier Trono Addgene #12260 pMB80 (R26-CreER) Dr. Tyler Jacks Addgene #12168 pmScarlet-i_C1 Dr. Dorus Gadella Addgene #85044 pLKO-PGK-rtTA-P2A-Cre This paper n/a pLKO-PGK-rtTA-P2A-Cre-SBE-NLS-mScarlet-I Taniguchi et al., 2020 n/a pLKO-PGK-rtTA-SBE-NLS-mScarlet-I-P2A-CreER This paper n/a Software and algorithms Fiji https://fiji.sc/ GraphPad Prism 9 GraphPad Software, LLC www.graphpad.com Other The Vevo2100 High-Resolution Micro-Imaging System FUJIFILM VisualSonics VS-20047 Nanoject III Auto-nanoliter injector Drummond Scientific Company 3-000-207 Open in a separate window Key resources table

Techniques: Recombinant, Imaging, Software

Figure 1. The LIM-peptidase region of DAR4 functions as an integrated decoy (A) Diagram of known protein motifs of DAR4 and mutant and chimeric forms used to test hypersensitive responses. TIR, Toll, and interleukin-1 receptor ho- mology regions; NB, NB-ARC nucleotide-binding; ARC, APAF-1,R proteins, and CED-4 domains; LRR, leucine-rich-repeat domain; LIM, LIN11, Isl1, and MEC3 domains; each Zn finger of the two LIM domains is represented; PEP, zinc-metallopeptidase region with AHEMMHA-EE active site; GST, glutathione S-transferase; GFP, green fluorescent protein. Red letters indicate the positions of mutated amino acid codons. C1340Y is in the fourth Zn-finger of the LIM repeats. H1488, H1492, E1508, and E1509 are in the HEMMA and EE motifs of the peptidase active site. The diagram is not to scale. Figure S2 shows the amino acid sequences and motifs of the DA1 family. (B and C) Hypersensitive responses in N. tabacum leaves were assessed at 4 days post-infection. Images represent typical samples of three replicates. DAR4 and variants in (A) were co-expressed as 3HA-tagged proteins with (B) or without (C) CSA1. Quantified HR responses29 are shown by the stacked bars are color coded to show the percentage of each cell death scale (0–5) in triplicated samples. Yellow represents the presence of HR, and green represents the absence of HR. (D) Immunoblots showing expression levels of 3HA-tagged CSA1 and CHS3/DAR4 variants in leaf infection assays in the left panel and dar4-LPGFP in the right immunoblot. 3HA-DA1 proteins were detected by HRP-coupled anti-HA antibodies, and GFP-tagged dar4-LPGFP was detected with HRP-coupled anti-GFP antibodies. To accommodate large differences in expression of 3HA-DAR4 deletions and mutants, different loadings on separate gels were electrophoresed in order to detect protein bands.

Journal: Cell host & microbe

Article Title: The integrated LIM-peptidase domain of the CSA1-CHS3/DAR4 paired immune receptor detects changes in DA1 peptidase inhibitors in Arabidopsis.

doi: 10.1016/j.chom.2023.04.009

Figure Lengend Snippet: Figure 1. The LIM-peptidase region of DAR4 functions as an integrated decoy (A) Diagram of known protein motifs of DAR4 and mutant and chimeric forms used to test hypersensitive responses. TIR, Toll, and interleukin-1 receptor ho- mology regions; NB, NB-ARC nucleotide-binding; ARC, APAF-1,R proteins, and CED-4 domains; LRR, leucine-rich-repeat domain; LIM, LIN11, Isl1, and MEC3 domains; each Zn finger of the two LIM domains is represented; PEP, zinc-metallopeptidase region with AHEMMHA-EE active site; GST, glutathione S-transferase; GFP, green fluorescent protein. Red letters indicate the positions of mutated amino acid codons. C1340Y is in the fourth Zn-finger of the LIM repeats. H1488, H1492, E1508, and E1509 are in the HEMMA and EE motifs of the peptidase active site. The diagram is not to scale. Figure S2 shows the amino acid sequences and motifs of the DA1 family. (B and C) Hypersensitive responses in N. tabacum leaves were assessed at 4 days post-infection. Images represent typical samples of three replicates. DAR4 and variants in (A) were co-expressed as 3HA-tagged proteins with (B) or without (C) CSA1. Quantified HR responses29 are shown by the stacked bars are color coded to show the percentage of each cell death scale (0–5) in triplicated samples. Yellow represents the presence of HR, and green represents the absence of HR. (D) Immunoblots showing expression levels of 3HA-tagged CSA1 and CHS3/DAR4 variants in leaf infection assays in the left panel and dar4-LPGFP in the right immunoblot. 3HA-DA1 proteins were detected by HRP-coupled anti-HA antibodies, and GFP-tagged dar4-LPGFP was detected with HRP-coupled anti-GFP antibodies. To accommodate large differences in expression of 3HA-DAR4 deletions and mutants, different loadings on separate gels were electrophoresed in order to detect protein bands.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 35S::3HA-dar4-pep This paper Addgene 200567 35S::3HA-dar4-EEII This paper Addgene 200568 35S::3HA-dar4-LP(GST) This paper Addgene 200569 35S::3HA-dar4-LP(GFP) This paper Addgene 200570 35S::3HA-DAR4-LP(DA1) This paper Addgene 200571 35S::3HA-DAR4-LP(DA1)-2d This paper Addgene 200572 35S::3HA-DAR4-LP(DAR1) This paper Addgene 200573 35S::3HA-DAR4-LP(DAR2) This paper Addgene 200574 35S::3HA-DAR4-LP(DAR3) This paper Addgene 200575 35S::3HA-DAR4-LP(DAR5) This paper Addgene 200576 35S::3HA-DAR4-LP(DAR6) This paper Addgene 200577 35S::3HA-DAR4-LP(DAR7) This paper Addgene 200578 35S::3HA-da1-pep Dong et al.23 Addgene 190759 35S::3HA-da1-L(DAR4) This paper Addgene 200579 35S::3HA-da1-L1(DAR4) This paper Addgene 200580 35S::3HA-da1-L2(DAR4) This paper Addgene 200581 35S::3HA-da1-L3(DAR4) This paper Addgene 200582 35S::3HA-da1-L4(DAR4) This paper Addgene 200583 35S::3HA-da1-P(DAR4) This paper Addgene 200584 35S::3HA-da1-P1(DAR4) This paper Addgene 200585 35S::3HA-da1-P2(DAR4) This paper Addgene 200586 35S::3HA-da1-P3(DAR4) This paper Addgene 200587 35S::3HA-da1-P2+3(DAR4) This paper Addgene 200588 35S::3HA-da1-LP(DAR4) This paper Addgene 200589 35S::DA1-GFP This paper Addgene 200590 35S::BB-3FLAG Dong et al.23 N/A 35S::DAR3-GFP This paper Addgene 200591 35S::DAR7-GFP This paper Addgene 200592 35S::dar3-LP(DA1)-GFP This paper Addgene 200593 35S::dar3-LP(DAR4)-GFP This paper Addgene 200594 35S::DAR4 aa1127-1613-GFP This paper Addgene 200595 35S::3HA-DAR4 aa1127-1613 This paper Addgene 200596 35S::3HA-DAR3 This paper Addgene 200597 35S::DA1-3FLAG Dong et al.23 N/A 35S::DAR1-3FLAG This paper Addgene 200598 35S::DAR2-3FLAG This paper Addgene 200599 35S::DAR3-3FLAG This paper Addgene 200600 35S::GFP Dong et al.23 Addgene 200600 35S::TIR(CSA1)-GFP This paper Addgene 200601 35S::TIR(DAR4)-GFP This paper Addgene 200602 DA1-pETnT Dong et al.23 N/A da1-LP(DAR4)-pETnT This paper Addgene 200603 da1-L(DAR4)-pETnT This paper Addgene 200604 da1-P(DAR4)-pETnT This paper Addgene 200605 DA2-pET24a Dong et al.23 N/A Software and algorithms MEGA-X Version X https://www.megasoftware.net/ e3 Cell Host & Microbe 31, 949–961.e1–e5, June 14, 2023

Techniques: Mutagenesis, Binding Assay, Infection, Western Blot, Expressing

Figure 3. Peptidase activities of LIM-peptidase regions of DAR4 in chimeric DA1 proteins (A) Diagram of chimeric 3HA-DA1 proteins used to test peptidase activities. Lilac, DA1 regions; blue, CHS3/DAR4 regions. The domain names are described in the legend to Figure 1. The diagram is not to scale. + shows BB-3FLAG cleavage; –, no cleavage; nt, not tested. (B) Immunoblots of BB-3FLAG cleavage reactions in transfected Arabidopsis da1dar1 mesophyll protoplasts using 3HA-DA1 chimeric proteins described in (A). 3HA-DA1 is a positive control and the 3HA-DA1 peptidase mutant (3HA-DA1 pep) is a negative control. BB-3FLAG proteins were detected by anti-FLAG-HRP antibodies. 3HA-DA1 proteins were detected by anti-HA-HRP antibodies. The positions of full-length (black arrow) and cleaved (red arrow) BB-FLAG proteins are indicated. The upper blot was split to remove a duplicated 3HA-DA1 + BB-3FLAG loading sample. All samples are from the same immunoblot exposed for the same time.

Journal: Cell host & microbe

Article Title: The integrated LIM-peptidase domain of the CSA1-CHS3/DAR4 paired immune receptor detects changes in DA1 peptidase inhibitors in Arabidopsis.

doi: 10.1016/j.chom.2023.04.009

Figure Lengend Snippet: Figure 3. Peptidase activities of LIM-peptidase regions of DAR4 in chimeric DA1 proteins (A) Diagram of chimeric 3HA-DA1 proteins used to test peptidase activities. Lilac, DA1 regions; blue, CHS3/DAR4 regions. The domain names are described in the legend to Figure 1. The diagram is not to scale. + shows BB-3FLAG cleavage; –, no cleavage; nt, not tested. (B) Immunoblots of BB-3FLAG cleavage reactions in transfected Arabidopsis da1dar1 mesophyll protoplasts using 3HA-DA1 chimeric proteins described in (A). 3HA-DA1 is a positive control and the 3HA-DA1 peptidase mutant (3HA-DA1 pep) is a negative control. BB-3FLAG proteins were detected by anti-FLAG-HRP antibodies. 3HA-DA1 proteins were detected by anti-HA-HRP antibodies. The positions of full-length (black arrow) and cleaved (red arrow) BB-FLAG proteins are indicated. The upper blot was split to remove a duplicated 3HA-DA1 + BB-3FLAG loading sample. All samples are from the same immunoblot exposed for the same time.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 35S::3HA-dar4-pep This paper Addgene 200567 35S::3HA-dar4-EEII This paper Addgene 200568 35S::3HA-dar4-LP(GST) This paper Addgene 200569 35S::3HA-dar4-LP(GFP) This paper Addgene 200570 35S::3HA-DAR4-LP(DA1) This paper Addgene 200571 35S::3HA-DAR4-LP(DA1)-2d This paper Addgene 200572 35S::3HA-DAR4-LP(DAR1) This paper Addgene 200573 35S::3HA-DAR4-LP(DAR2) This paper Addgene 200574 35S::3HA-DAR4-LP(DAR3) This paper Addgene 200575 35S::3HA-DAR4-LP(DAR5) This paper Addgene 200576 35S::3HA-DAR4-LP(DAR6) This paper Addgene 200577 35S::3HA-DAR4-LP(DAR7) This paper Addgene 200578 35S::3HA-da1-pep Dong et al.23 Addgene 190759 35S::3HA-da1-L(DAR4) This paper Addgene 200579 35S::3HA-da1-L1(DAR4) This paper Addgene 200580 35S::3HA-da1-L2(DAR4) This paper Addgene 200581 35S::3HA-da1-L3(DAR4) This paper Addgene 200582 35S::3HA-da1-L4(DAR4) This paper Addgene 200583 35S::3HA-da1-P(DAR4) This paper Addgene 200584 35S::3HA-da1-P1(DAR4) This paper Addgene 200585 35S::3HA-da1-P2(DAR4) This paper Addgene 200586 35S::3HA-da1-P3(DAR4) This paper Addgene 200587 35S::3HA-da1-P2+3(DAR4) This paper Addgene 200588 35S::3HA-da1-LP(DAR4) This paper Addgene 200589 35S::DA1-GFP This paper Addgene 200590 35S::BB-3FLAG Dong et al.23 N/A 35S::DAR3-GFP This paper Addgene 200591 35S::DAR7-GFP This paper Addgene 200592 35S::dar3-LP(DA1)-GFP This paper Addgene 200593 35S::dar3-LP(DAR4)-GFP This paper Addgene 200594 35S::DAR4 aa1127-1613-GFP This paper Addgene 200595 35S::3HA-DAR4 aa1127-1613 This paper Addgene 200596 35S::3HA-DAR3 This paper Addgene 200597 35S::DA1-3FLAG Dong et al.23 N/A 35S::DAR1-3FLAG This paper Addgene 200598 35S::DAR2-3FLAG This paper Addgene 200599 35S::DAR3-3FLAG This paper Addgene 200600 35S::GFP Dong et al.23 Addgene 200600 35S::TIR(CSA1)-GFP This paper Addgene 200601 35S::TIR(DAR4)-GFP This paper Addgene 200602 DA1-pETnT Dong et al.23 N/A da1-LP(DAR4)-pETnT This paper Addgene 200603 da1-L(DAR4)-pETnT This paper Addgene 200604 da1-P(DAR4)-pETnT This paper Addgene 200605 DA2-pET24a Dong et al.23 N/A Software and algorithms MEGA-X Version X https://www.megasoftware.net/ e3 Cell Host & Microbe 31, 949–961.e1–e5, June 14, 2023

Techniques: Western Blot, Transfection, Positive Control, Mutagenesis, Negative Control

Figure 4. DA1 and DAR1 facilitate growth of bacterial and oomycete pathogens in Arabidopsis (A) Pseudomonas syringae Pst DC3000 growth on Arabidopsis Col-0 da1 and dar1 knockout lines and 35S::DA1 overexpressing lines. CFUs, colony-forming units per mg fresh weight of inoculated leaves. The eds1-2 mutant was used as a standard susceptible control. (B) Hyaloperonospora arabidopsidis (Hpa) race Noco2 growth on Arabidopsis Col-0 da1 and dar1 knockout lines and 35S::DA1 over-expressing lines. FW, fresh weight of spores in infected plants. The eds1-2 mutant was used as a susceptible control. (C) Time course of Albugo candida AcEm2 growth on Arabidopsis Ws-2 and backcross lines with Col-0 da1dar1. wt-10 and wt-44 contain wt DA1 and DAR1 loci from Ws-2 in BC7F2, while da1-ko1dar1-1_Ws-2 contains homozygous Col-0 da1dar1 mutants in BC7F2. Infection progress was measured using the ratio of pathogen DNA to host DNA levels. dpi, days post-infection. Statistical significance was at *p < 0.01 based on a two-tailed Student’s t test. Comparisons of pathogen levels were those in Ws-2 at 0 dpi. Error bars represent SD of the mean. (D) Image of Albugo candida AcEm2 growth on leaves of Ws-2 and backcrossed lines at 12 dpi leaves. The white areas are Em2, and the arrow indicates a small amount of pathogen growth on the da1-ko1dar1-1_Ws-2 backcrossed line.

Journal: Cell host & microbe

Article Title: The integrated LIM-peptidase domain of the CSA1-CHS3/DAR4 paired immune receptor detects changes in DA1 peptidase inhibitors in Arabidopsis.

doi: 10.1016/j.chom.2023.04.009

Figure Lengend Snippet: Figure 4. DA1 and DAR1 facilitate growth of bacterial and oomycete pathogens in Arabidopsis (A) Pseudomonas syringae Pst DC3000 growth on Arabidopsis Col-0 da1 and dar1 knockout lines and 35S::DA1 overexpressing lines. CFUs, colony-forming units per mg fresh weight of inoculated leaves. The eds1-2 mutant was used as a standard susceptible control. (B) Hyaloperonospora arabidopsidis (Hpa) race Noco2 growth on Arabidopsis Col-0 da1 and dar1 knockout lines and 35S::DA1 over-expressing lines. FW, fresh weight of spores in infected plants. The eds1-2 mutant was used as a susceptible control. (C) Time course of Albugo candida AcEm2 growth on Arabidopsis Ws-2 and backcross lines with Col-0 da1dar1. wt-10 and wt-44 contain wt DA1 and DAR1 loci from Ws-2 in BC7F2, while da1-ko1dar1-1_Ws-2 contains homozygous Col-0 da1dar1 mutants in BC7F2. Infection progress was measured using the ratio of pathogen DNA to host DNA levels. dpi, days post-infection. Statistical significance was at *p < 0.01 based on a two-tailed Student’s t test. Comparisons of pathogen levels were those in Ws-2 at 0 dpi. Error bars represent SD of the mean. (D) Image of Albugo candida AcEm2 growth on leaves of Ws-2 and backcrossed lines at 12 dpi leaves. The white areas are Em2, and the arrow indicates a small amount of pathogen growth on the da1-ko1dar1-1_Ws-2 backcrossed line.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 35S::3HA-dar4-pep This paper Addgene 200567 35S::3HA-dar4-EEII This paper Addgene 200568 35S::3HA-dar4-LP(GST) This paper Addgene 200569 35S::3HA-dar4-LP(GFP) This paper Addgene 200570 35S::3HA-DAR4-LP(DA1) This paper Addgene 200571 35S::3HA-DAR4-LP(DA1)-2d This paper Addgene 200572 35S::3HA-DAR4-LP(DAR1) This paper Addgene 200573 35S::3HA-DAR4-LP(DAR2) This paper Addgene 200574 35S::3HA-DAR4-LP(DAR3) This paper Addgene 200575 35S::3HA-DAR4-LP(DAR5) This paper Addgene 200576 35S::3HA-DAR4-LP(DAR6) This paper Addgene 200577 35S::3HA-DAR4-LP(DAR7) This paper Addgene 200578 35S::3HA-da1-pep Dong et al.23 Addgene 190759 35S::3HA-da1-L(DAR4) This paper Addgene 200579 35S::3HA-da1-L1(DAR4) This paper Addgene 200580 35S::3HA-da1-L2(DAR4) This paper Addgene 200581 35S::3HA-da1-L3(DAR4) This paper Addgene 200582 35S::3HA-da1-L4(DAR4) This paper Addgene 200583 35S::3HA-da1-P(DAR4) This paper Addgene 200584 35S::3HA-da1-P1(DAR4) This paper Addgene 200585 35S::3HA-da1-P2(DAR4) This paper Addgene 200586 35S::3HA-da1-P3(DAR4) This paper Addgene 200587 35S::3HA-da1-P2+3(DAR4) This paper Addgene 200588 35S::3HA-da1-LP(DAR4) This paper Addgene 200589 35S::DA1-GFP This paper Addgene 200590 35S::BB-3FLAG Dong et al.23 N/A 35S::DAR3-GFP This paper Addgene 200591 35S::DAR7-GFP This paper Addgene 200592 35S::dar3-LP(DA1)-GFP This paper Addgene 200593 35S::dar3-LP(DAR4)-GFP This paper Addgene 200594 35S::DAR4 aa1127-1613-GFP This paper Addgene 200595 35S::3HA-DAR4 aa1127-1613 This paper Addgene 200596 35S::3HA-DAR3 This paper Addgene 200597 35S::DA1-3FLAG Dong et al.23 N/A 35S::DAR1-3FLAG This paper Addgene 200598 35S::DAR2-3FLAG This paper Addgene 200599 35S::DAR3-3FLAG This paper Addgene 200600 35S::GFP Dong et al.23 Addgene 200600 35S::TIR(CSA1)-GFP This paper Addgene 200601 35S::TIR(DAR4)-GFP This paper Addgene 200602 DA1-pETnT Dong et al.23 N/A da1-LP(DAR4)-pETnT This paper Addgene 200603 da1-L(DAR4)-pETnT This paper Addgene 200604 da1-P(DAR4)-pETnT This paper Addgene 200605 DA2-pET24a Dong et al.23 N/A Software and algorithms MEGA-X Version X https://www.megasoftware.net/ e3 Cell Host & Microbe 31, 949–961.e1–e5, June 14, 2023

Techniques: Knock-Out, Mutagenesis, Control, Expressing, Infection, Two Tailed Test

Figure 6. DAR3 and DAR7 family members inhibit DA1 peptidase activity Immunoblots of Col-0 da1-ko1dar1-1 transfected Arabidopsis mesophyll protoplasts. The numbers above each lane show the fold levels of 10 mg units of transfected DNA 35S::GFP was used to balance total DNA levels to 60 mg per transfection. The red arrows indicate cleaved BB-3FLAG while the black arrows indicate intact BB-3FLAG. (A) Co-immunoprecipitation of 3HA-DAR3 and DA1-, DAR1-, and DAR2-3FLAG. (B) Progressive inhibition of 3HA-DA1 peptidase activity on BB-3FLAG by increasing levels of DAR3-GFP. (C) Increased levels of inactive DA1-peptidase mutant-GFP do not inhibit 3HA-DA1 peptidase activity on BB-3FLAG. (D) DAR7-GFP also progressively inhibits 3HA-DA1 peptidase activity on BB-3-FLAG. (E) Progressive reduction of 3HA-DA1 peptidase activity on BB-3FLAG by DAR3-GFP containing the LIM-peptidase region of DAR4. (F) DAR3-GFP containing the LIM-peptidase region of DA1 does not affect 3HA-DA1 peptidase activity on BB-3FLAG. (G) The LIM-Peptidase domain of DAR4 (DAR41127-1613 – GFP) does not reduce 3HA-DA1- mediated cleavage of BB-3FLAG. (H) 3HA-DA1 protein levels are not affected by co-expression with DAR3-GFP and LIM-Peptidase-GFP proteins.

Journal: Cell host & microbe

Article Title: The integrated LIM-peptidase domain of the CSA1-CHS3/DAR4 paired immune receptor detects changes in DA1 peptidase inhibitors in Arabidopsis.

doi: 10.1016/j.chom.2023.04.009

Figure Lengend Snippet: Figure 6. DAR3 and DAR7 family members inhibit DA1 peptidase activity Immunoblots of Col-0 da1-ko1dar1-1 transfected Arabidopsis mesophyll protoplasts. The numbers above each lane show the fold levels of 10 mg units of transfected DNA 35S::GFP was used to balance total DNA levels to 60 mg per transfection. The red arrows indicate cleaved BB-3FLAG while the black arrows indicate intact BB-3FLAG. (A) Co-immunoprecipitation of 3HA-DAR3 and DA1-, DAR1-, and DAR2-3FLAG. (B) Progressive inhibition of 3HA-DA1 peptidase activity on BB-3FLAG by increasing levels of DAR3-GFP. (C) Increased levels of inactive DA1-peptidase mutant-GFP do not inhibit 3HA-DA1 peptidase activity on BB-3FLAG. (D) DAR7-GFP also progressively inhibits 3HA-DA1 peptidase activity on BB-3-FLAG. (E) Progressive reduction of 3HA-DA1 peptidase activity on BB-3FLAG by DAR3-GFP containing the LIM-peptidase region of DAR4. (F) DAR3-GFP containing the LIM-peptidase region of DA1 does not affect 3HA-DA1 peptidase activity on BB-3FLAG. (G) The LIM-Peptidase domain of DAR4 (DAR41127-1613 – GFP) does not reduce 3HA-DA1- mediated cleavage of BB-3FLAG. (H) 3HA-DA1 protein levels are not affected by co-expression with DAR3-GFP and LIM-Peptidase-GFP proteins.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 35S::3HA-dar4-pep This paper Addgene 200567 35S::3HA-dar4-EEII This paper Addgene 200568 35S::3HA-dar4-LP(GST) This paper Addgene 200569 35S::3HA-dar4-LP(GFP) This paper Addgene 200570 35S::3HA-DAR4-LP(DA1) This paper Addgene 200571 35S::3HA-DAR4-LP(DA1)-2d This paper Addgene 200572 35S::3HA-DAR4-LP(DAR1) This paper Addgene 200573 35S::3HA-DAR4-LP(DAR2) This paper Addgene 200574 35S::3HA-DAR4-LP(DAR3) This paper Addgene 200575 35S::3HA-DAR4-LP(DAR5) This paper Addgene 200576 35S::3HA-DAR4-LP(DAR6) This paper Addgene 200577 35S::3HA-DAR4-LP(DAR7) This paper Addgene 200578 35S::3HA-da1-pep Dong et al.23 Addgene 190759 35S::3HA-da1-L(DAR4) This paper Addgene 200579 35S::3HA-da1-L1(DAR4) This paper Addgene 200580 35S::3HA-da1-L2(DAR4) This paper Addgene 200581 35S::3HA-da1-L3(DAR4) This paper Addgene 200582 35S::3HA-da1-L4(DAR4) This paper Addgene 200583 35S::3HA-da1-P(DAR4) This paper Addgene 200584 35S::3HA-da1-P1(DAR4) This paper Addgene 200585 35S::3HA-da1-P2(DAR4) This paper Addgene 200586 35S::3HA-da1-P3(DAR4) This paper Addgene 200587 35S::3HA-da1-P2+3(DAR4) This paper Addgene 200588 35S::3HA-da1-LP(DAR4) This paper Addgene 200589 35S::DA1-GFP This paper Addgene 200590 35S::BB-3FLAG Dong et al.23 N/A 35S::DAR3-GFP This paper Addgene 200591 35S::DAR7-GFP This paper Addgene 200592 35S::dar3-LP(DA1)-GFP This paper Addgene 200593 35S::dar3-LP(DAR4)-GFP This paper Addgene 200594 35S::DAR4 aa1127-1613-GFP This paper Addgene 200595 35S::3HA-DAR4 aa1127-1613 This paper Addgene 200596 35S::3HA-DAR3 This paper Addgene 200597 35S::DA1-3FLAG Dong et al.23 N/A 35S::DAR1-3FLAG This paper Addgene 200598 35S::DAR2-3FLAG This paper Addgene 200599 35S::DAR3-3FLAG This paper Addgene 200600 35S::GFP Dong et al.23 Addgene 200600 35S::TIR(CSA1)-GFP This paper Addgene 200601 35S::TIR(DAR4)-GFP This paper Addgene 200602 DA1-pETnT Dong et al.23 N/A da1-LP(DAR4)-pETnT This paper Addgene 200603 da1-L(DAR4)-pETnT This paper Addgene 200604 da1-P(DAR4)-pETnT This paper Addgene 200605 DA2-pET24a Dong et al.23 N/A Software and algorithms MEGA-X Version X https://www.megasoftware.net/ e3 Cell Host & Microbe 31, 949–961.e1–e5, June 14, 2023

Techniques: Activity Assay, Western Blot, Transfection, Immunoprecipitation, Inhibition, Mutagenesis, Expressing

Journal: eLife

Article Title: Predicted glycosyltransferases promote development and prevent spurious cell clumping in the choanoflagellate S. rosetta

doi: 10.7554/eLife.41482

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pEFl5'-Actin3'::pac-P2A-couscous-mTFP, , , this paper , Addgene ID NK698 , Parent vector: pEFl5'- Actin3'::pac-P2A-mTFP ; couscous inserted using Gibson assembly.

Techniques: Recombinant, Marker, Plasmid Preparation

Figure 1. CCPG1 Is an LIR Motif-Containing Interactor of Human ATG8 Orthologs (A) Schematic of CCPG1 structure (NTD, N-terminal amino acids 1–230; TM, transmembrane anchor). (B) GST or GST fusions of ATG8 orthologs (LC3B, LC3C, and GABARAP) were used in affinity precipitation (AP) of transfected myc-CCPG1 from HEK293 cells. (C) GST or GST-GABARAP (mtLDS, LIR-docking site mutant) were used in AP of transfected myc-CCPG1 NTD from HEK293 cells.

Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 1. CCPG1 Is an LIR Motif-Containing Interactor of Human ATG8 Orthologs (A) Schematic of CCPG1 structure (NTD, N-terminal amino acids 1–230; TM, transmembrane anchor). (B) GST or GST fusions of ATG8 orthologs (LC3B, LC3C, and GABARAP) were used in affinity precipitation (AP) of transfected myc-CCPG1 from HEK293 cells. (C) GST or GST-GABARAP (mtLDS, LIR-docking site mutant) were used in AP of transfected myc-CCPG1 NTD from HEK293 cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: Transfection, Mutagenesis

Figure 2. CCPG1 Is a FIP200-Interacting Protein (A) A549 NTAP (FLAG-HA)-CCPG1 cells were immunoprecipitated for tagged CCPG1 using anti-HA antibody and immunoprecipitates subjected to LC-MS/MS and CompPASS analysis (see the STAR Methods and Table S1). Interacting proteins at a cut-off of WDN score 0.8 are shown here. (B) A549 cells stably expressing NTAP empty vector () or NTAP-CCPG1 (+) were immunoprecipitated for tagged CCPG1 with anti-FLAG beads and im- munoblotted for indicated proteins. (C) A549 cells were EBSS starved or left untreated for 1 hr, prior to lysis and endogenous immunoprecipitation of CCPG1 and subsequent immunoblotting (IgG, negative control IgG). (D) HEK293 cells were transfected with FLAG-FIP200 and indicated variants of full-length (FL) GFP-CCPG1 (DNTD, amino acids 231–757). Immunoprecipitation was performed with GFP-Trap and immunoblotting performed with indicated antibodies. (E) Recombinant FIP200 was incubated with either glutathione Sepharose beads alone, or with pre-purified GST or GST-CCPG1 NTD bound beads. Affinity precipitation (AP) followed by immunoblotting was then performed to assess direct interaction. See also Figure S1 and Table S1.

Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 2. CCPG1 Is a FIP200-Interacting Protein (A) A549 NTAP (FLAG-HA)-CCPG1 cells were immunoprecipitated for tagged CCPG1 using anti-HA antibody and immunoprecipitates subjected to LC-MS/MS and CompPASS analysis (see the STAR Methods and Table S1). Interacting proteins at a cut-off of WDN score 0.8 are shown here. (B) A549 cells stably expressing NTAP empty vector () or NTAP-CCPG1 (+) were immunoprecipitated for tagged CCPG1 with anti-FLAG beads and im- munoblotted for indicated proteins. (C) A549 cells were EBSS starved or left untreated for 1 hr, prior to lysis and endogenous immunoprecipitation of CCPG1 and subsequent immunoblotting (IgG, negative control IgG). (D) HEK293 cells were transfected with FLAG-FIP200 and indicated variants of full-length (FL) GFP-CCPG1 (DNTD, amino acids 231–757). Immunoprecipitation was performed with GFP-Trap and immunoblotting performed with indicated antibodies. (E) Recombinant FIP200 was incubated with either glutathione Sepharose beads alone, or with pre-purified GST or GST-CCPG1 NTD bound beads. Affinity precipitation (AP) followed by immunoblotting was then performed to assess direct interaction. See also Figure S1 and Table S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Stable Transfection, Expressing, Plasmid Preparation, Lysis, Western Blot, Negative Control, Transfection, Recombinant, Incubation

Figure 3. Identification of a Linear Peptide Motif in CCPG1 for Binding to FIP200 C-Terminal Region (A) A 15-mer peptide array (peptides 1–55) was probed with recombinant FIP200. Bound FIP200 was detected by indirect immunodetection. Peptide sequences corresponding to binding regions A–C are shown below the array. (B and C) HEK293 cells were transfected with FLAG-FIP200 and indicated myc-tagged deletions or truncations of CCPG1 NTD prior to anti-myc immunopre- cipitation and immunoblotting (EV, empty vector). (D) Sequence alignment of the region from amino acids 97 to 118 of human CCPG1 against vertebrate orthologs (upper) or of regions amino acids 99–113 and 17– 31 of human CCPG1 (lower). Conserved S/T and acidic residues are blue, hydrophobic residues are red. Asterisks indicate evolutionary conservation of residues. Black boxes indicate residues identical between FIR1 and FIR2. (legend continued on next page)

Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 3. Identification of a Linear Peptide Motif in CCPG1 for Binding to FIP200 C-Terminal Region (A) A 15-mer peptide array (peptides 1–55) was probed with recombinant FIP200. Bound FIP200 was detected by indirect immunodetection. Peptide sequences corresponding to binding regions A–C are shown below the array. (B and C) HEK293 cells were transfected with FLAG-FIP200 and indicated myc-tagged deletions or truncations of CCPG1 NTD prior to anti-myc immunopre- cipitation and immunoblotting (EV, empty vector). (D) Sequence alignment of the region from amino acids 97 to 118 of human CCPG1 against vertebrate orthologs (upper) or of regions amino acids 99–113 and 17– 31 of human CCPG1 (lower). Conserved S/T and acidic residues are blue, hydrophobic residues are red. Asterisks indicate evolutionary conservation of residues. Black boxes indicate residues identical between FIR1 and FIR2. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: Binding Assay, Peptide Microarray, Recombinant, Immunodetection, Transfection, Western Blot, Plasmid Preparation, Sequencing

Figure 4. CCPG1 Is Recruited into Autophagosomes from the ER (A) A549 cells were transfected with siCtrl or siCCPG1 and, at 24 hr post-transfection, either left untreated or starved for 1 hr in EBSS, then stained for endogenous CCPG1. Cells with CCPG1 foci were scored (n = 3, ± SEM, *p < 0.05, two-tailed paired sample t tests). Scale bar, 20 mm. (legend continued on next page)

Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 4. CCPG1 Is Recruited into Autophagosomes from the ER (A) A549 cells were transfected with siCtrl or siCCPG1 and, at 24 hr post-transfection, either left untreated or starved for 1 hr in EBSS, then stained for endogenous CCPG1. Cells with CCPG1 foci were scored (n = 3, ± SEM, *p < 0.05, two-tailed paired sample t tests). Scale bar, 20 mm. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: Transfection, Staining, Two Tailed Test

Figure 5. CCPG1 Is a UPR-Inducible Gene that Remodels the ER (A) A549 cells were treated with indicated ER stressors for 16 hr (Tun, tunicamycin, 2.5 mg/mL and Thaps, thapsigargin, 0.5 mM). qRT-PCR was performed for CCPG1 (n = 3, ± SEM, *p < 0.05, one-way ANOVA followed by Tukey’s post-hoc test). (B) HeLa cells were treated with indicated ER stressors (DTT, 0.5 or 2 mM, and Tun at 1 or 2.5 mg/mL, or Thaps at 0.5 mM) for 16 hr and then immunoblotted.

Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 5. CCPG1 Is a UPR-Inducible Gene that Remodels the ER (A) A549 cells were treated with indicated ER stressors for 16 hr (Tun, tunicamycin, 2.5 mg/mL and Thaps, thapsigargin, 0.5 mM). qRT-PCR was performed for CCPG1 (n = 3, ± SEM, *p < 0.05, one-way ANOVA followed by Tukey’s post-hoc test). (B) HeLa cells were treated with indicated ER stressors (DTT, 0.5 or 2 mM, and Tun at 1 or 2.5 mg/mL, or Thaps at 0.5 mM) for 16 hr and then immunoblotted.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: Quantitative RT-PCR

Figure 6. Defective Proteostasis in the Pancreas of Ccpg1 Hypomorphic Mice (A and B) Whole pancreata from littermate 6-week-old WT (+/+) or Ccpg1 hypomorphic (GT/GT) mice were immunoblotted for CCPG1 or subjected to RNA extraction and qRT-PCR for Ccpg1 (n = 3 pairs, ± SEM, ***p < 0.001, two-tailed t test). (C and D) Fifty mg of whole pancreata from littermate pairs of 6-week-old WT and Ccpg1 hypomorphic mice were homogenized in SDS. Insoluble protein was pelleted, washed and extracted in 8 M urea +10 mM DTT. Pellet samples were normalized according to protein concentration in the soluble fraction and subjected to label-free LC-MS/MS quantification. A median absolute deviation analysis is presented as a heatmap here to show species changing significantly between pairs of mice (pairs joined by connecting brackets). Secretory enzymes are in red, ER luminal chaperones/oxidoreductases are in blue. (E and F) Detergent soluble and insoluble samples prepared as above were immunoblotted and ratios of insoluble to soluble protein species obtained via densitometry (n = 3 pairs, ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t tests). See also Figure S5 and Table S2.

Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 6. Defective Proteostasis in the Pancreas of Ccpg1 Hypomorphic Mice (A and B) Whole pancreata from littermate 6-week-old WT (+/+) or Ccpg1 hypomorphic (GT/GT) mice were immunoblotted for CCPG1 or subjected to RNA extraction and qRT-PCR for Ccpg1 (n = 3 pairs, ± SEM, ***p < 0.001, two-tailed t test). (C and D) Fifty mg of whole pancreata from littermate pairs of 6-week-old WT and Ccpg1 hypomorphic mice were homogenized in SDS. Insoluble protein was pelleted, washed and extracted in 8 M urea +10 mM DTT. Pellet samples were normalized according to protein concentration in the soluble fraction and subjected to label-free LC-MS/MS quantification. A median absolute deviation analysis is presented as a heatmap here to show species changing significantly between pairs of mice (pairs joined by connecting brackets). Secretory enzymes are in red, ER luminal chaperones/oxidoreductases are in blue. (E and F) Detergent soluble and insoluble samples prepared as above were immunoblotted and ratios of insoluble to soluble protein species obtained via densitometry (n = 3 pairs, ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t tests). See also Figure S5 and Table S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: RNA Extraction, Quantitative RT-PCR, Two Tailed Test, Protein Concentration, Liquid Chromatography with Mass Spectroscopy

Figure 7. Loss of Cell Polarization and ER Homeostasis, and Consequent Tissue Injury, in Ccpg1 Hypomorphic Exocrine Pancreata (A) The acinar unit of the exocrine pancreas. Polarized acinar cells secrete condensed enzyme (zymogen) granules into ducts from their apical stores. These enzymes are initially synthesized in the expansive rough ER (rER), which occupies the basolateral regions of the cell. (B) CARS imaging or immunohistochemical staining for the ER (protein disulfide isomerase, PDI) in pancreatic tissue from 6-week-old littermate WT (+/+) or Ccpg1 hypomorphic (GT/GT) mice. Punctate CARS signals indicate protein or lipid inclusions. Scale bars, 20 mm. (C) Transmission electron microscopy (TEM) of pancreata from 6-week-old littermate pairs. Scale bar, 5 mm. Analysis of percent cytosolic area occupied by osmophilic protein granules was performed in ImageJ (n = 4 pairs, ± SEM, *p < 0.05, two-tailed t test). (D) High magnification TEM of a Ccpg1 hypomorphic mouse reveals that the rER is distended and many supernumerary inclusions are in fact intracisternal granule-like structures (arrows in zoomed inset). Scale bar, 1 mm.

Journal: Developmental cell

Article Title: CCPG1 Is a Non-canonical Autophagy Cargo Receptor Essential for ER-Phagy and Pancreatic ER Proteostasis.

doi: 10.1016/j.devcel.2017.11.024

Figure Lengend Snippet: Figure 7. Loss of Cell Polarization and ER Homeostasis, and Consequent Tissue Injury, in Ccpg1 Hypomorphic Exocrine Pancreata (A) The acinar unit of the exocrine pancreas. Polarized acinar cells secrete condensed enzyme (zymogen) granules into ducts from their apical stores. These enzymes are initially synthesized in the expansive rough ER (rER), which occupies the basolateral regions of the cell. (B) CARS imaging or immunohistochemical staining for the ER (protein disulfide isomerase, PDI) in pancreatic tissue from 6-week-old littermate WT (+/+) or Ccpg1 hypomorphic (GT/GT) mice. Punctate CARS signals indicate protein or lipid inclusions. Scale bars, 20 mm. (C) Transmission electron microscopy (TEM) of pancreata from 6-week-old littermate pairs. Scale bar, 5 mm. Analysis of percent cytosolic area occupied by osmophilic protein granules was performed in ImageJ (n = 4 pairs, ± SEM, *p < 0.05, two-tailed t test). (D) High magnification TEM of a Ccpg1 hypomorphic mouse reveals that the rER is distended and many supernumerary inclusions are in fact intracisternal granule-like structures (arrows in zoomed inset). Scale bar, 1 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER pdcDNA 6x myc CCPG1 Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1 S22A D23A I24A E25A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR2 S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pdcDNA 6x myc CCPG1 NTD CCPG1 Human CCPG1 1-230 This paper N/A pdcDNA 6x myc CCPG1 NTD Human CCPG1 1-230 with internal deletions or truncated from C-terminus, as indicated in main text This paper N/A pdcDNA FLAG-FIP200 Human FIP200 1279-1594 This paper N/A pEGFP-C1 Clontech # 6084-1 pEGFP-CCPG1 CCPG1 Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 mtLIR + mtFIR1+2 W14A I17A S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pEGFP-CCPG1 NTD Human CCPG11-230 This paper N/A pEGFP-CCPG1 DNTD Human CCPG1 231-757 This paper N/A pmCherry-ER-3 A gift from Michael Davidson, MagLab, USA Addgene plasmid # 55041 pMXs-puro GFP-DFCP1 A gift from Noboru Mizushima, Tokyo medical and dental University, Japan (Itakura and Mizushima, 2010) Addgene plasmid # 38269 pRevTRE EGFP Clontech # 6137-1 pRevTRE GFP-CCPG1 Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtLIR W14A I17A Human CCPG1 1-757 This paper N/A pRevTRE GFP-CCPG1 mtFIR1+2 S22A D23A I24A E25A S104A D105A I106A L109A Human CCPG1 1-757 This paper N/A pSpCas9(BB)-2A-Puro (PX45) v2.0 A gift from Feng Zhang, Broad Institute, USA (Ran et al., 2013) Addgene plasmid # 62988 (Continued on next page) Developmental Cell 44, 217–232.e1–e11, January 22, 2018 e5

Techniques: Synthesized, Imaging, Immunohistochemical staining, Staining, Transmission Assay, Electron Microscopy, Two Tailed Test