Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: Overexpression of TRIM38 inhibits TNFα- and IL-1β–triggered signaling. (A) Effects of TRIM38 on TNFα- and IL-1β–triggered NF-κB activation in HEK293 cells. HEK293 cells (1 × 105) were transfected with the NF-κB luciferase plasmid (0.01 μg) and an HA-TRIM38 plasmid (0.2 or 0.4 μg). Twenty hours after transfection, cells were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) or left untreated for 10 h before luciferase assays were performed. Expression of transfected TRIM38 in each unstimulated sample was examined by immunoblot analysis. (B) Effects of TRIM38 on TNFα- and IL-1β–triggered NF-κB activation in HCT116 and HeLa cells. The experiments were performed as in A. (C) Effects of TRIM38 on IFNγ-induced activation of the IRF1 promoter. The experiments were performed as in A except that the IRF1 promoter reporter plasmid was used and transfected cells were treated with IFNγ (100 ng/mL). (D) Effects of TRIM38 on TNFα- and IL-1β–induced transcription of TNFA, IL-6, and IL-8 genes. HEK293 cells were transduced with either an empty vector or an HA-TRIM38 plasmid to establish stable cell lines. Cells (4 × 105) from both stable cell lines were treated with TNFα or IL-1β for the indicated times, and then total RNA was prepared for qPCR analysis. Expression of TRIM38 in the stable cell lines was examined by immunoblot analysis (Right). (E) Effects of TRIM38 on TNFα- and IL-1β–induced cytokine of TNFα, IL-6, and IL-8. HEK293 cells were transduced with either an empty vector or an HA-TRIM38 plasmid to establish stable cell lines. Cells (4 × 105) from both stable cell lines were treated with TNFα or IL-1β for the indicated times, and then the medium was collected for ELISA analysis. (F) Effects of TRIM38 on IFNγ-induced transcription of IRF1 gene. Cells (4 × 105) were left untreated or treated with IFNγ (100 ng/mL) for the indicated times, and total RNA was extracted for qPCR analysis. Graphs show mean ± SD; n = 3. *P < 0.05; **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Over Expression, Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Western Blot, Transduction, Stable Transfection, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: Knockdown or knockout of TRIM38 potentiates TNFα- and IL-1β–triggered signaling. (A) Efficiencies of TRIM38-RNAi plasmids on TRIM38 levels. (Upper) HEK293 cells (4 × 105) were transfected with expression plasmids for TRIM38-Flag and HA-β-actin (0.1 μg each) and the indicated RNAi plasmids (1 μg each). Twenty-four hours after transfection, cell lysates were analyzed by immunoblot with anti-Flag or anti-HA. (Lower) HEK293 cells (1 × 107) were transfected with control or the indicated TRIM38-RNAi plasmids (10 μg each) for 36 h. Cell lysates were analyzed by immunoblot with anti-TRIM38 or anti–β-actin. (B) Effects of TRIM38-RNAi on TNFα- and IL-1β–triggered NF-κB activation in HEK293 and HeLa cells. The cells (1 × 105) were transfected with RNAi plasmids (1 μg each) along with the NF-κB reporter plasmid (0.01 μg). Thirty-six hours after transfection, cells were left untreated or treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for 10 h before luciferase assays were performed. (C) Effects of TRIM38-RNAi on IFNγ-induced IRF1 promoter activation. Reporter assays were performed as in B except that cells were transfected with IRF1 promoter reporter plasmid and treated with IFNγ (100 ng/mL). (D) Effects of TRIM38 deficiency on TNFα and IL-1β–induced transcription of TNFA, IL-6, and IL-8 genes. The indicated cells (4 × 105) were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for the indicated times, and then total RNA was extracted for qPCR analysis. (E) Effects of TRIM38 deficiency on TNFα and IL-1β–induced cytokine production. The indicated cells (4 × 105) were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for the indicated times, and then the medium was collected for ELISA analysis. (F) TRIM38 deficiency potentiates TNFα-triggered MAPK activation. The indicated cells (1 × 107) were left untreated or treated with TNFα (10 ng/mL) for the indicated times. Cells were lyzed and immunoblot analysis was performed with the indicated antibodies. (G) TRIM38 deficiency potentiates IL-1β–triggered MAPK activation. The experiments were performed as in E, except that cells were treated with IL-1β (10 ng/mL). Graphs show mean ± SD; n = 3. *P < 0.05; **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Knockdown, Knock-Out, Transfection, Expressing, Western Blot, Control, Activation Assay, Plasmid Preparation, Luciferase, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: TRIM38 interacts with and destabilizes TAB2 through its C-terminal PRY-SPRY domain. (A) TRIM38 interacts with TAB2 and TAB3 in mammalian overexpression system. HEK293 cells (1 × 107) were transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies. (B) Endogenous TRIM38 interacts with TAB2/3. HEK293 cells (3 × 107) were left untreated or treated with TNFα (Left) or IL-1β (Right) for the indicated times. Endogenous coimmunoprecipitation and immunoblots were performed with the indicated antibodies. (C) TRIM38 specifically destabilizes TAB2/3. HEK293 (4 × 105) cells were transfected with the indicated plasmids for 24 h, and then immunoblots were performed with the indicated antibodies. (D) Effects of TRIM38 truncation mutants on destabilization of TAB2. HEK293 (4 × 105) cells were transfected with the indicated plasmids for 24 h before immunoblots were performed with the indicated antibodies. (E) Analysis of TRIM38 expression in TRIM38−/− cells stably transduced with an empty vector (II), TRIM38-Flag (III), TRIM38(63-465)-Flag (IV), or TRIM38(290-465)-Flag (V), respectively and in TRIM38+/+ cells stably transduced with an empty vector (I). Cells (1 × 107) (I, II, III, IV, V) were harvested and lysed. Immunoblot analysis was performed with the indicated antibodies. (F) Reconstitution of TRIM38 or TRIM38 mutant (63–465) into TRIM38-deficient cells leads to down-regulation of TAB2. Cells (1 × 107) (I, II, III, IV, V) were left untreated or treated with TNFα or IL-1β for the indicated times. Immunoblot analysis was performed with the indicated antibodies.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Over Expression, Transfection, Western Blot, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: TRIM38 mediates lysosomal degradation of TAB2. (A) Effects of inhibitors on TRIM38-mediated destabilization of TAB2. HEK293 cells (4 × 105) were transfected with the indicated plasmids. Fourteen hours after transfection, the cells were treated with the indicated inhibitors for 6 h before immunoblot analysis was performed. (B) Effects of NH4Cl and MG132 on down-regulation of TAB2 triggered by TNFα and IL-1β. HEK293 (1 × 107) cells were treated with NH4Cl or MG132 for 4 h and then further treated with TNFα and IL-1β for 2 h before immunoblot analysis was performed. (C) TRIM38 promotes translocation of TAB2 to the lysosome. HEK293 cells (1 × 105) were transfected with Cherry-TAB2 and GFP-LAMP1 (Left) or CFP-TRIM38 (Right). Twenty hours after transfection, cells were fixed with 4% (wt/vol) paraformaldehyde and subjected for confocal microscopy. (D) Effect of TRIM38 deficiency on TNFα- or IL-1β–induced colocalization of TAB2 with the lysosomes. TRIM38+/+, TRIM38−/−, or TRIM38−/− cells reconstituted with the PRY-SPRY domain (1 × 105) were transfected with GFP-TAB2. Twenty hours after transfection, cells were stained with Red Lysotracker (200 nM) for 2 h and treated with TNFα (20 ng/mL) or IL-1β (20 ng/mL) for 1 h and then fixed with 4% (wt/vol) paraformaldehyde and subjected to confocal microscopy. A random 10 cells in each sample were used for calculating the colocalization dots that were normalized to the total lysosome-red dots. Graphs show mean ± SD; n = 3. **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Transfection, Western Blot, Translocation Assay, Confocal Microscopy, Staining

Journal: Neurobiology of learning and memory

Article Title: Rescue of Synaptic Plasticity and Spatial Learning Deficits in the Hippocampus of Homer1 Knockout Mice by Recombinant Adeno-Associated Viral Gene Delivery of Homer1c

doi: 10.1016/j.nlm.2011.08.009

Figure Lengend Snippet: Subcellular distribution of Homer1c and its interacting partners, mGluR1α and mGluR5, in CA1. (A) Panels show immunohistochemistry of wildtype and H1-KO animals injected with either high dose H1c, low dose H1c or GFP. LM-WT mice express Homer1c at a lower level than CB57/BL6, as previously observed by Western blot analysis (Figure 1E). Labeling with GFAP reveals activated astrocytes in the stratum radiatum of KO+H1c animals, occurring both at the high and (to a lesser extent) the low dose of H1c. (B) Immunofluorescence images illustrating co-localization of mGluR5 (green, left column) and GFAP (red, middle column) in the hippocampus of the experimental animals shown. Double immunofluorescence is indicated in yellow in the merged image (right column). SP, stratum pyramidale; SO, stratum oriens-alveus; SR, stratum radiatum.

Article Snippet: Sections were then incubated overnight in primary antibody with 1% normal serum, washed, incubated in 1:300 biotinylated secondary IgG (goat anti-mouse for mGluR1α, goat anti-rabbit for mGluR5 and GFAP, or rabbit anti-goat for Homer 1c; Vector, Burlingame, CA) for 3 hours, followed by 1 hour in avidin-biotin complex (Standard Elite kit; Vector).

Techniques: Immunohistochemistry, Injection, Western Blot, Labeling, Immunofluorescence

Journal:

Article Title: Interferon Regulatory Factor 7 Mediates Activation of Tap-2 by Epstein-Barr Virus Latent Membrane Protein 1

doi: 10.1128/JVI.75.1.341-350.2001

Figure Lengend Snippet: Activation of Tap-2 promoter reporter construct by IRF-7. (A) Schematic diagrams of Tap-2 reporter constructs. Top line, Tap-2 genomic region; open rectangle, ISRE. A 573-bp fragment from the Tap-2 promoter region was cloned; the ISRE sequence and mutations are shown. (B) Schematic diagrams of various IRF-7 expression plasmids. IRF-7A, -7B, and -7C are splicing variants of IRF-7. IRF-7DN lacks the DNA-binding domain. (C) Activation of Tap-2 reporter construct by IRF-7 in B cells. Akata cells were transfected with the reporter construct Tap2-CAT together with vector pcDNA-3 (column 1) or expression plasmids for IRF-1 (column 2), IRF-2 (column 3), IRF-3 (column 4), ICSBP (column 5), IRF-7A (column 6), IRF-7B (column 7), IRF-7DN (column 8), or LMP-1 (column 9). Columns 10 to 16, pcLMP-1 was cotransfected with IRF-1, IRF-2, IRF-3, ICSBP, IRF-7A, IRF-7B, and IRF-7DN, respectively. (D) Mutations in ISRE abolish activation by IRF-7. Akata cells were transfected with the reporter construct mTap2-CAT and pcDNA-3 (column 1) or IRF-7A (column 2) or IRF-7A plus pcLMP-1 (column 3) or IRF-1 expression plasmid (column 4). CAT assay results were normalized to β-galactosidase activity. CAT activity is expressed relative to the vector control. Standard deviations are shown.

Article Snippet: The IRF-1 (C-20) and IRF-2 (C-19) antibodies were purchased from Santa Cruz Biotechnology, Inc. LMP-1 monoclonal antibody CS1-4 was purchased from Dako.

Techniques: Activation Assay, Construct, Clone Assay, Sequencing, Expressing, Binding Assay, Transfection, Plasmid Preparation, Activity Assay

Journal: Journal of dairy science

Article Title: Caveolin 1 in bovine liver is associated with fatty acid-induced lipid accumulation and the ER unfolded protein response: role in fatty liver development.

doi: 10.3168/jds.2024-25349

Figure Lengend Snippet: Figure 2. Hepatic endoplasmic reticulum (ER) stress and lipogenic state in dairy cows with fatty liver. (A) Representative Western blots of phosphorylated protein kinase RNA-like ER kinase (p-PERK), phosphorylated inositol requiring protein-1α (p-IRE1α), inositol requiring protein-1α (IRE1α), protein kinase RNA-like ER kinase (PERK), 78-kDa glucose-regulated protein (GRP78/Bip), activating transcription factor-6 (ATF6), C/EBP-homologous protein (CHOP), and β-actin. (B) The ratios of p-PERK/PERK, p-IRE1α/IRE1α, GRP78, ATF6, and CHOP in healthy (n = 6) and fatty liver (n = 6) cows. (C) Relative mRNA expression levels of GRP78, ATF4, and sXBP1 in healthy (n = 6) and fatty liver (n = 6) cows. (D) Representative Western blots of Acetyl-CoA carboxylase 1 (ACC1), Sterol regulatory element binding protein 1c (SREBP-1c), and peroxisome proliferator-activated receptor γ (PPARγ). (E) The ratios of ACC1, SREBP-1c, PPARγ in healthy (n = 6) and fatty liver (n = 6) cows. (F) Relative mRNA expression levels of Fatty Acid Synthase (FASN), Acetyl Coenzyme A Carboxylase Alpha (ACACA), and Sterol regulatory element binding protein 1 (SREBP1) in healthy (n = 6) and fatty liver (n = 6) cows. The data of the control were used to normalize other treatments. Comparisons among groups were calculated using unpaired t-tests and expressed as means ± SEM.

Article Snippet: The blocked membranes were incubated overnight at 4°C with specific antibodies for FLAG (1:5000, 66008–4-Ig; ProteinTech), Phospho-PERK (1:1000, 29546–1-AP; ProteinTech), PERK (1:1000, 20582–1-AP; ProteinTech), Phospho-IRE1α (1:1000, AF7150; Affinity Biosciences), IRE1α (1:1000, 27528–1-AP; ProteinTech), GRP78/BIP (1:1000, 11587–1-AP; ProteinTech), ATF6 (1:1000, 24169–1-AP; ProteinTech), CHOP (1:1000, 15204–1-AP; ProteinTech), Caveolin-1 (1:1000, 1:100; bs-1453R, Bioss), SREBP-1c (1:1,000, NB100–2215; Novus Biologicals, Littleton, CO), ACC1 (1:1,000, 3662; Cell Signaling, Danvers, MA), PPARγ (1:1,000, A0270, Abclonal), and β-actin (internal control; 1:1,000, sc-47778; Santa Cruz Biotechnology) and treated for 30 min at RT with HRP-conjugated secondary antibodies (3:5,000; Beyotime Biotechnology).

Techniques: Western Blot, Expressing, Binding Assay, Control

Journal: Journal of dairy science

Article Title: Caveolin 1 in bovine liver is associated with fatty acid-induced lipid accumulation and the ER unfolded protein response: role in fatty liver development.

doi: 10.3168/jds.2024-25349

Figure Lengend Snippet: Figure 3. Effects of free fatty acid (FFA) treatment on endoplasmic reticulum (ER) stress induction and lipogenic gene expression in calf primary hepatocytes. For the time-course analysis experiment, calf primary hepatocytes were treated with FFA (1.2 mM) for 1, 3, 6, and 12 h, respectively. (A) Identification of cytokeratin-18 immunofluorescence. (B) Representative Western blots of phosphorylated protein kinase RNA-like ER ki- nase (p-PERK), phosphorylated inositol requiring protein-1α (p-IRE1α), inositol requiring protein-1α (IRE1α), protein kinase RNA-like ER kinase (PERK), 78-kDa glucose-regulated protein (GRP78/Bip), activating transcription factor-6 (ATF6), C/EBP-homologous protein (CHOP), caveolin-1, and β-actin. (C) The ratios of (i) Caveolin 1, (ii) p-PERK/PERK, (iii) p-IRE1α/IRE1α, (iv) GRP78, (v) ATF6, and (vi) CHOP were quantified in calf primary hepatocytes following NEFA treatment at different time points. (D) Representative Western blots of Acetyl-CoA carboxylase 1 (ACC1), Sterol regulatory element binding protein 1c (SREBP-1c), and peroxisome proliferator-activated receptor γ (PPARγ). (E) The ratios of (i) ACC1, (ii) SREBP-1c, (iii) PPARγ were quantified in calf primary hepatocytes following NEFA treatment at different time points. (F) immunofluorescence results of lipid droplets (n = 3 per group, scale bar = 25 μm). Comparisons among groups were calculated using a one-way ANOVA with subsequent Bonferroni correction. All data are presented as means ± SEM *P < 0.05, **P < 0.01 compared with the 1 h group.

Article Snippet: The blocked membranes were incubated overnight at 4°C with specific antibodies for FLAG (1:5000, 66008–4-Ig; ProteinTech), Phospho-PERK (1:1000, 29546–1-AP; ProteinTech), PERK (1:1000, 20582–1-AP; ProteinTech), Phospho-IRE1α (1:1000, AF7150; Affinity Biosciences), IRE1α (1:1000, 27528–1-AP; ProteinTech), GRP78/BIP (1:1000, 11587–1-AP; ProteinTech), ATF6 (1:1000, 24169–1-AP; ProteinTech), CHOP (1:1000, 15204–1-AP; ProteinTech), Caveolin-1 (1:1000, 1:100; bs-1453R, Bioss), SREBP-1c (1:1,000, NB100–2215; Novus Biologicals, Littleton, CO), ACC1 (1:1,000, 3662; Cell Signaling, Danvers, MA), PPARγ (1:1,000, A0270, Abclonal), and β-actin (internal control; 1:1,000, sc-47778; Santa Cruz Biotechnology) and treated for 30 min at RT with HRP-conjugated secondary antibodies (3:5,000; Beyotime Biotechnology).

Techniques: Gene Expression, Immunofluorescence, Western Blot, Binding Assay

Journal: Journal of dairy science

Article Title: Caveolin 1 in bovine liver is associated with fatty acid-induced lipid accumulation and the ER unfolded protein response: role in fatty liver development.

doi: 10.3168/jds.2024-25349

Figure Lengend Snippet: Figure 4. The effect of caveolin 1 overexpression on free fatty acid (FFA)-induced endoplasmic reticulum (ER) stress induction and lipogenic gene and protein expression in calf primary hepatocytes. Cells were transfected with pCMV-T7-MCS-3 × FLAG-WPRE-Neo plasmid (pc-DNA) or pCMV-CAV1(cattle)-3 × FLAG-Neo (pc-CAV1) overexpression plasmid by lipofectamine 2000 for 36 h followed by FFA for another 12 h. (A) Relative hepatocytic mRNA levels of caveolin 1. (B) Representative Western blots of phosphorylated protein kinase RNA-like ER kinase (p-PERK), phosphorylated inositol requiring protein-1α (p-IRE1α), inositol requiring protein-1α (IRE1α), protein kinase RNA-like ER kinase (PERK), 78-kDa glucose-regulated protein (GRP78/Bip), activating transcription factor-6 (ATF6), cleaved-ATF6, C/EBP-homologous protein (CHOP), caveolin-1, and β-actin. (C) The ratios of (i) Caveolin 1, (ii) p-PERK/PERK, (iii) p-IRE1α/IRE1α, (iv) GRP78, (v) ATF6, (vi) cleaved-ATF6, and (vii) CHOP were quantified in calf primary hepatocytes. (D) Representative Western blots of Acetyl-CoA carboxylase 1 (ACC1), Sterol regulatory element binding protein 1c (SREBP-1c), and peroxisome proliferator-activated receptor γ (PPARγ). (E) The ratios of (i) ACC1, (ii) SREBP-1c, (iii) PPARγ were quantified in calf primary hepatocytes. (F) Relative mRNA expression levels of GRP78, ATF4, and sXBP1. (G) Relative mRNA expression levels of Fatty Acid Synthase (FASN), Acetyl Coenzyme A Carboxylase Alpha (ACACA), and Sterol regulatory element binding protein 1 (SREBP1) in calf primary hepatocytes. (H) immunofluorescence results of lipid droplets (n = 3 per group, scale bar = 25 μm). Data were analyzed with one-way ANOVA with subsequent Bonferroni correction and presented as the means ± SEM (n = 3). Different superscript lowercase letters in bar charts represent significant difference (P < 0.05).

Article Snippet: The blocked membranes were incubated overnight at 4°C with specific antibodies for FLAG (1:5000, 66008–4-Ig; ProteinTech), Phospho-PERK (1:1000, 29546–1-AP; ProteinTech), PERK (1:1000, 20582–1-AP; ProteinTech), Phospho-IRE1α (1:1000, AF7150; Affinity Biosciences), IRE1α (1:1000, 27528–1-AP; ProteinTech), GRP78/BIP (1:1000, 11587–1-AP; ProteinTech), ATF6 (1:1000, 24169–1-AP; ProteinTech), CHOP (1:1000, 15204–1-AP; ProteinTech), Caveolin-1 (1:1000, 1:100; bs-1453R, Bioss), SREBP-1c (1:1,000, NB100–2215; Novus Biologicals, Littleton, CO), ACC1 (1:1,000, 3662; Cell Signaling, Danvers, MA), PPARγ (1:1,000, A0270, Abclonal), and β-actin (internal control; 1:1,000, sc-47778; Santa Cruz Biotechnology) and treated for 30 min at RT with HRP-conjugated secondary antibodies (3:5,000; Beyotime Biotechnology).

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot, Binding Assay, Immunofluorescence

Journal: Journal of dairy science

Article Title: Caveolin 1 in bovine liver is associated with fatty acid-induced lipid accumulation and the ER unfolded protein response: role in fatty liver development.

doi: 10.3168/jds.2024-25349

Figure Lengend Snippet: Figure 5. The effect of silencing caveolin 1 on free fatty acid (FFA)-induced endoplasmic reticulum (ER) stress induction and lipogenic gene and protein expression in calf primary hepatocytes. Cells were treated with caveolin 1 transient transfection (siCAV1) for 36 h, and incubated with 1.2 mM FFA for another 12 h. (A) Relative hepatocytic mRNA levels of caveolin 1. (B) Representative Western blots of phosphorylated protein kinase RNA-like ER kinase (p-PERK), phosphorylated inositol requiring protein-1α (p-IRE1α), inositol requiring protein-1α (IRE1α), protein kinase RNA-like ER kinase (PERK), 78-kDa glucose-regulated protein (GRP78/Bip), activating transcription factor-6 (ATF6), cleaved-ATF6, C/EBP- homologous protein (CHOP), caveolin-1, and β-actin. (C) The ratios of (i) caveolin 1, (ii) p-PERK/PERK, (iii) p-IRE1α/IRE1α, (iv) GRP78, (v) ATF6, (vi) cleaved-ATF6, and (vii) CHOP were quantified in calf primary hepatocytes. (D) Representative Western blots of Acetyl-CoA carboxylase 1 (ACC1), Sterol regulatory element binding protein 1c (SREBP-1c), and peroxisome proliferator-activated receptor γ (PPARγ). (E) The ratios of (i) ACC1, (ii) SREBP-1c, (iii) PPARγ were quantified in calf primary hepatocytes. (F) Relative mRNA expression levels of GRP78, ATF4, and sXBP1. (G) Relative mRNA expression levels of Fatty Acid Synthase (FASN), Acetyl Coenzyme A Carboxylase Alpha (ACACA), and Sterol regulatory ele- ment binding protein 1 (SREBP1). (H) immunofluorescence results of lipid droplets (n = 3 per group, scale bar = 25 μm). Data were analyzed with one-way ANOVA with subsequent Bonferroni correction and presented as the means ± SEM (n = 3). Different superscript lowercase letters in bar charts represent significant difference (P < 0.05).

Article Snippet: The blocked membranes were incubated overnight at 4°C with specific antibodies for FLAG (1:5000, 66008–4-Ig; ProteinTech), Phospho-PERK (1:1000, 29546–1-AP; ProteinTech), PERK (1:1000, 20582–1-AP; ProteinTech), Phospho-IRE1α (1:1000, AF7150; Affinity Biosciences), IRE1α (1:1000, 27528–1-AP; ProteinTech), GRP78/BIP (1:1000, 11587–1-AP; ProteinTech), ATF6 (1:1000, 24169–1-AP; ProteinTech), CHOP (1:1000, 15204–1-AP; ProteinTech), Caveolin-1 (1:1000, 1:100; bs-1453R, Bioss), SREBP-1c (1:1,000, NB100–2215; Novus Biologicals, Littleton, CO), ACC1 (1:1,000, 3662; Cell Signaling, Danvers, MA), PPARγ (1:1,000, A0270, Abclonal), and β-actin (internal control; 1:1,000, sc-47778; Santa Cruz Biotechnology) and treated for 30 min at RT with HRP-conjugated secondary antibodies (3:5,000; Beyotime Biotechnology).

Techniques: Expressing, Transfection, Incubation, Western Blot, Binding Assay, Immunofluorescence

Journal: Journal of dairy science

Article Title: Caveolin 1 in bovine liver is associated with fatty acid-induced lipid accumulation and the ER unfolded protein response: role in fatty liver development.

doi: 10.3168/jds.2024-25349

Figure Lengend Snippet: Figure 6. Effects of caveolin 1 overexpression, tunicamycin (TM), or both, on endoplasmic reticulum (ER) stress induction and lipogenic gene expression in calf primary hepatocytes. Hepatocytes were cultured in the presence or absence of pc-CAV1 for 24 h, cells were treated with 0.5 μg/ mL TM for 24 h. (A) Representative Western blots of phosphorylated protein kinase RNA-like ER kinase (p-PERK), phosphorylated inositol requir- ing protein-1α (p-IRE1α), inositol requiring protein-1α (IRE1α), protein kinase RNA-like ER kinase (PERK), 78-kDa glucose-regulated protein (GRP78/Bip), activating transcription factor-6 (ATF6), cleaved-ATF6, C/EBP-homologous protein (CHOP), caveolin-1, and β-actin. (B) The ratios of (i) caveolin 1, (ii) p-PERK/PERK, (iii) p-IRE1α/IRE1α, (iv) GRP78, (v) ATF6, (vi) cleaved-ATF6, and (vii) CHOP were quantified in calf primary hepatocytes. (C) Representative Western blots of Acetyl-CoA carboxylase 1 (ACC1), Sterol regulatory element binding protein 1c (SREBP-1c), and peroxisome proliferator-activated receptor γ (PPARγ). (D) The ratios of (i) ACC1, (ii) SREBP-1c, (iii) PPARγ were quantified in calf primary hepatocytes. (E) Relative mRNA expression levels of GRP78, ATF4, and sXBP1. (F) Relative mRNA expression levels of Fatty Acid Synthase (FASN), Acetyl Coenzyme A Carboxylase Alpha (ACACA), and Sterol regulatory element binding protein 1 (SREBP1). (G) Co-IP analysis to assay the interaction between Flag-caveolin 1 and UPR indicators in calf primary hepatocytes. Calf primary hepatocytes were transfected with pCMV- CAV1(cattle)-3 × FLAG-Neo (pc-CAV1) overexpression plasmid for 48 h. Data were analyzed with one-way ANOVA with subsequent Bonferroni correction and presented as the means ± SEM (n = 3). Different superscript lowercase letters in bar charts represent significant difference (P < 0.05).

Article Snippet: The blocked membranes were incubated overnight at 4°C with specific antibodies for FLAG (1:5000, 66008–4-Ig; ProteinTech), Phospho-PERK (1:1000, 29546–1-AP; ProteinTech), PERK (1:1000, 20582–1-AP; ProteinTech), Phospho-IRE1α (1:1000, AF7150; Affinity Biosciences), IRE1α (1:1000, 27528–1-AP; ProteinTech), GRP78/BIP (1:1000, 11587–1-AP; ProteinTech), ATF6 (1:1000, 24169–1-AP; ProteinTech), CHOP (1:1000, 15204–1-AP; ProteinTech), Caveolin-1 (1:1000, 1:100; bs-1453R, Bioss), SREBP-1c (1:1,000, NB100–2215; Novus Biologicals, Littleton, CO), ACC1 (1:1,000, 3662; Cell Signaling, Danvers, MA), PPARγ (1:1,000, A0270, Abclonal), and β-actin (internal control; 1:1,000, sc-47778; Santa Cruz Biotechnology) and treated for 30 min at RT with HRP-conjugated secondary antibodies (3:5,000; Beyotime Biotechnology).

Techniques: Over Expression, Gene Expression, Cell Culture, Western Blot, Binding Assay, Expressing, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation