Journal: Nature structural & molecular biology

Article Title: CNPY2 is a Key Initiator of the PERK-CHOP Pathway of the Unfolded Protein Response

doi: 10.1038/nsmb.3458

Figure Lengend Snippet: ( a ) Immunoblotting of WT, Cnpy2 Het and KO liver and kidney tissues at 8 weeks of age injected with DMSO or Tu for 24 h. ( b ) Hematoxylin and eosin (H&E) and Oil Red O staining for liver sections from indicated mice. Scale bar: 100 μm. ( c ) Serum ALT levels of WT, Cnpy2 Het and KO animals measured at 24 h post Tu treatment. ( d and e ) Cell viability ( d ) and immunoblotting ( e ) of WT, Cnpy2 Het and KO primary hepatocytes treated with DMSO, TG or Tu for 16 h. Data are shown as the mean ± s.d. of two ( a-c , WT: n=12; Het: n=11; KO: n=11 mice) or three ( d and e ) independent experiments. * P < 0.05, ** P < 0.01 by Student’s t -test ( c and d ). Uncropped images for a and e are shown in .

Article Snippet: Antibodies against CNPY2 (Cat.# 14635-1-AP), Myc (Cat.# 16286-1-AP) and (Cat.# 11587-1-AP) were purchased from Proteintech.

Techniques: Western Blot, Injection, Staining

Journal: Nature structural & molecular biology

Article Title: CNPY2 is a Key Initiator of the PERK-CHOP Pathway of the Unfolded Protein Response

doi: 10.1038/nsmb.3458

Figure Lengend Snippet: ( a,b ) Body weight at 10 weeks ( a ) and photograph of liver lobes ( b ) of Cnpy2 -sufficent (Het) and –deficient (KO) male mice given high fat diet. * P < 0.05 by Student’s t -test( c ) H&E and Oil Red O staining for Cnpy2 Het and KO liver sections. (Scale bar: 100 μm). ( d ) Immunoblotting of Cnpy2 Het and KO liver lysates at 10 weeks after giving high fat diet. ( e,f ) Serum ALT ( e ) and triglycerides ( f ) levels were measured. * P <0.05; ** P <0.01 by Student’s t -test. All data are shown as the mean ± s.d. of two independent experiments ( a-e ). n= 10 mice per group. Uncropped blot/gel images of d are shown in .

Article Snippet: Antibodies against CNPY2 (Cat.# 14635-1-AP), Myc (Cat.# 16286-1-AP) and (Cat.# 11587-1-AP) were purchased from Proteintech.

Techniques: Staining, Western Blot

Journal: Nature structural & molecular biology

Article Title: CNPY2 is a Key Initiator of the PERK-CHOP Pathway of the Unfolded Protein Response

doi: 10.1038/nsmb.3458

Figure Lengend Snippet: ( a ) Sequential immunoprecipitation (IP) of Myc-grp78 and FLAG-CNPY2-co-overexpressing HEK293 cells treated with or without Tu, followed by immunoblotting for the indicated proteins. ( b ) co-IP and immunoblotting (as in a ) of HEK293 cells co-transfected with FLAG-CNPY2 and Myc-PERK vectors. ( c ) In vitro binding assay of purified 6X His/T7-tagged PERK-LD and His-tagged CNPY2 assessed by immunoblotting. 6X His-tagged protein (6X-His) was used as a control. HC: immunoglobulin heavy chain. ( d ) Study of CNPY2-PERK interaction by isothermal titration calorimetry (ITC). Data shown are the mean ± s.d. of three independent experiments. ( e ) IP using anti-T7 antibody and immunoblotting of His/T7-tagged PERK-LD and recombinant grp78 proteins incubated in the presence or absence of His-tagged CNPY2 or bovine serum albumin (BSA). ( f ) PERK kinase assay performed in the presence or absence of CNPY2 using GST-eif2α, GST-eif2αS51A or GST as substrates. Phosphorylation was visualized using an autoradiography. “exp.” denotes exposure. Input proteins were verified by immunoblotting. HC: immunoglobulin heavy chain. Data are representative of three ( a-d ) or two ( e-f ) independent experiments. Uncropped blot/gel images of a-c and e,f are shown in .

Article Snippet: Antibodies against CNPY2 (Cat.# 14635-1-AP), Myc (Cat.# 16286-1-AP) and (Cat.# 11587-1-AP) were purchased from Proteintech.

Techniques: Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Transfection, In Vitro, Binding Assay, Purification, Control, Isothermal Titration Calorimetry, Recombinant, Incubation, Perk Kinase Assay, Phospho-proteomics, Autoradiography

Journal: Nature structural & molecular biology

Article Title: CNPY2 is a Key Initiator of the PERK-CHOP Pathway of the Unfolded Protein Response

doi: 10.1038/nsmb.3458

Figure Lengend Snippet: ( a ) Homogeneous His/T7-tagged PERK-LD and His-tagged CNPY2 WT or Cys mutant 3C-A (C28/37/86A) were incubated, followed by IP using anti-T7 antibody. Indicated proteins were immunoblotted with anti-His antibody. ( b ) Study of the interaction between PERK and CNPY2 mutants by ITC. Data are mean ± s.d. of three independent experiments. ( c ) In vitro PERK kinase assay (as in ) was performed in the presence or absence of CNPY2 WT or CNPY2 3C-A mutant protein. Phosphorylation of GST-eif2α was visualized using an autoradiography. Input proteins are shown in the Coomassie blue-stained gel. ( d ) Motility of WT CNPY2 and its Cys mutants on reducing and non-reducing SDS-PAGE. Immunoblotting was performed using total cell lysates of MEFs expressing various forms of CNPY2. β-actin was used as a loading control. ( e ) Restoration of UPR by WT or CNPY2 mutants in CNPY2 KO MEFs cells treated with or without Tu for 6 h, and then immunoblotted for the indicated proteins. EV: Empty vector. Data are representative of three ( a,b and e ) or two ( c,d ) independent experiments. Uncropped blot/gel images of a and c-e are shown in .

Article Snippet: Antibodies against CNPY2 (Cat.# 14635-1-AP), Myc (Cat.# 16286-1-AP) and (Cat.# 11587-1-AP) were purchased from Proteintech.

Techniques: Mutagenesis, Incubation, In Vitro, Perk Kinase Assay, Phospho-proteomics, Autoradiography, Staining, SDS Page, Western Blot, Expressing, Control, Plasmid Preparation

Journal: Nature structural & molecular biology

Article Title: CNPY2 is a Key Initiator of the PERK-CHOP Pathway of the Unfolded Protein Response

doi: 10.1038/nsmb.3458

Figure Lengend Snippet: ( a ) Alignment of CHOP-responsive element found in the promoter regions of Cnpy2 , sarcoendoplasmic reticulum calcium ATPase1 (Human SERCA1; Murine Serca1) , carbonic anhydrase VI (Human CAVI ), chaperonins 60/10 (Human CPN60/10 ) and Tribbles pseudokinase 3 (Human TRB3 ; Murine Trb3 ) genes. The base positions of the consensus are boxed and indicated 5′→3′. Positions are relative to transcription initiation site or where indicated (asterisk) to initiation codon. ( b ) Oligonucleotide pull-down assay of RAW cells treated with DMSO or Tu for 16 h and. Immunoblotting of DNA-protein complexes in nuclear extracts (input) and on pull-down complexes (Oligo-bound). ( c ) Chromatin immunoprecipitation assay of Myc-tagged CHOP-expressing HEK293 cell lysates with anti-Myc antibodies or control antibodies, followed by PCR for Cnpy2 and Trb3 promoters. ( d ) Luciferase activity measured in HEK293 cells 48 h after transfection with Cnpy2 promoter-driven luciferase (Luc) reporter ( Cnpy2-Luc ) with or without CHOP-expression vector. Data shown is the mean ± s.d. of three independent experiments. * P < 0.05 by Student’s t -test. ( e ) Immunoblotting for the indicated proteins in the whole cell lysate of ( d ). Data are representative of three ( b-e ) independent experiments. Uncropped blot/gel images for b and e are shown in .

Article Snippet: Antibodies against CNPY2 (Cat.# 14635-1-AP), Myc (Cat.# 16286-1-AP) and (Cat.# 11587-1-AP) were purchased from Proteintech.

Techniques: Pull Down Assay, Western Blot, Chromatin Immunoprecipitation, Expressing, Control, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Nature structural & molecular biology

Article Title: CNPY2 is a Key Initiator of the PERK-CHOP Pathway of the Unfolded Protein Response

doi: 10.1038/nsmb.3458

Figure Lengend Snippet: Under non-ER stress conditions, grp78 binds to PERK and CNPY2, and thus maintains these proteins in an inactive state. In the presence of ER stress, accumulation of misfolded proteins or alterations in ER homeostasis causes grp78 to dissociate from both PERK and CNPY2 to perform its chaperone function for misfolded proteins. grp78 dissociation, along with CNPY2 binding to PERK, and perhaps other yet unidentified events collectively initiate the UPR. The activated PERK pathway increases CHOP expression, which in turn transcriptionally upregulates CNPY2 expression.

Article Snippet: Antibodies against CNPY2 (Cat.# 14635-1-AP), Myc (Cat.# 16286-1-AP) and (Cat.# 11587-1-AP) were purchased from Proteintech.

Techniques: Binding Assay, Expressing

Journal: iScience

Article Title: The microcephaly-associated protein YIPF5 differentially regulates ER export

doi: 10.1016/j.isci.2026.114791

Figure Lengend Snippet: YIPF5 regulates the secretome composition (A) Schematic representation of the experimental workflow for analyzing the glycoprotein secretome. MCF10A cells were metabolically labeled with the clickable sugar analog ManNAz, followed by a biotin-azide click reaction to tag glycoproteins ( n = 4). Secreted glycoproteins were concentrated from the culture medium, selectively purified, and identified using mass spectrometry. The image was created with BioRender.com . (B) Quantitative comparison of protein abundance in the secretome versus the total proteome of MCF10A cells. Proteins with secretion changes reflecting a similar change in total expression (“explained by proteome”) (q value ≤ 0.05) are shown in green. Proteins with increased or decreased secretion independent of total proteome changes are highlighted in red and blue, respectively. Gray (not affected) represents unchanged proteins. (C) Analysis of protein abundance changes in the secretome of YIPF5 KO versus control MCF10A cells (WT). Proteins significantly upregulated (red) or downregulated (blue) in the YIPF5 KO secretome are shown. Proteins with secretion changes explained by total proteome abundance are shown in green, while unaffected proteins are in gray. The vertical axis represents –log10(q value), and the horizontal axis represents log2(fold change). (D and E) Heatmap depicting proteins showing increased secretion in the YIPF5 KO secretome with a gene ontology (GO) annotation for the ER (GO: 0007029) and the Golgi apparatus (GO: 0005794) (D) or that contain a KDEL-ER retrieval sequence (E). (F) MCF10A WT and YIPF5 KO cells stably expressing the inducible ER-Ca 2+ sensor GCampER were analyzed using live fluorescence microscopy at identical settings. (G) Mean ± SD fluorescence intensity of GCampER was quantified in MCF10A WT and YIPF5 KO cells in 72 wells per cell line from n = 3 independent experiments. Unpaired t test, two-tailed p < 0.0001. (H) HeLa cells stably expressing shScramble or shRNAs against YIPF5 were transiently transfected with CNPY3-mCherry, supernatants were collected and cell lysates prepared after 24 h followed by western blot analysis using a CNPY3 antibody. A representative blot of n = 3 independent experiments is shown. For full size blot see C. (I) Quantification of CNPY3-secretion from (H). Displayed are arbitrary units normalized to the values of shScramble-expressing cells from n = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test, with ∗ indicating p < 0.05. (J) Analysis of protein abundance changes in the secretome of YIPF5 KO re-expressing YIPF5 I98S mutant versus control MCF10A cells (WT). Proteins significantly upregulated (red) or downregulated (blue) in the YIPF5 I98S secretome are shown ( n = 4). Proteins with secretion changes explained by total proteome abundance are denoted in green, while unaffected proteins are in gray. (K) Venn Diagram of protein abundance changes in YIPF5-KO cells and YIPF5-KO re-expressing YIPF5 I98S cells. Proteins with decreased (blue) or increased (red) secretome abundance are shown. See also and , , , , and .

Article Snippet: CNPY3 rabbit polyclonal , Proteintech , Cat# 15215-1-AP; RRID: AB_11182172.

Techniques: Metabolic Labelling, Labeling, Purification, Mass Spectrometry, Comparison, Quantitative Proteomics, Expressing, Control, Sequencing, Stable Transfection, Fluorescence, Microscopy, Two Tailed Test, Transfection, Western Blot, Mutagenesis