primary antibodies against atf6  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: MODULATION OF LIPID METABOLISM BY THAPSIGARGIN INHIBITS HEPATITIS C VIRUS INFECTION

doi: 10.64898/2026.03.02.709032

Figure Lengend Snippet: Huh7.5 cells were treated with DMSO or 10 µM IXA4 for 72 hours. (A) Cellular RNA was harvested and XBP1s (IRE1 pathway), CHOP (PERK pathway) and HERPUD1 (ATF6 pathway) mRNA expression was assessed by RT qPCR. (B) Expression of genes related to LD biogenesis ( FASN , DGAT1 and DGAT2 ) was assessed by RT-qPCR. (C) Huh7 cells were electroporated with 5 µg of HCV SGR RNA. Four hours-post electroporation, cells were treated with DMSO or 10 µM IXA4 for 72h and RNA replication was measured by luciferase expression. Data are expressed as a percentage of HCV replication in DMSO-treated samples. (D-E) Huh7.5 cells were pre-treated with DMSO or 0.05 µM Tg for 30 minutes, and subsequently infected with HCV at MOI 0.2 for 72h. (D) Cells were fixed and stained for cell nuclei (DAPI, blue) and LDs (BODIPY-493; green), HCV core (red), and assessed by confocal microscopy under 63x magnification. (E) Cellular supernatant was collected, and viral extracellular titer was assessed by focus forming assay. Graphs shown mean +/− SEM from 3 independent experiments. RT-qPCR was performed in technical triplicate per biological replicate. Confocal microscopy data are representative of at least three biological replicates with at least three technical replicates per condition. *p<0.05, **p<0.01, ***p<0.0005, ****p<0.0001.

Article Snippet: Primary antibodies against ATF6 (Cell Signaling Technology (CST) 8089, dilution 1:1000), IRE1a (CST 3294, dilution 1:1000), PERK (CST 5683, dilution 1:1000), and beta-actin (Abcam ab8226, dilution 1:5000), and secondary antibodies Licor IRDye-680 goat anti-mouse and Licor IRDye-800 were used for western blot analysis.

Techniques: Expressing, Quantitative RT-PCR, Electroporation, Luciferase, Infection, Staining, Confocal Microscopy, Focus Forming Assay

Journal: bioRxiv

Article Title: MODULATION OF LIPID METABOLISM BY THAPSIGARGIN INHIBITS HEPATITIS C VIRUS INFECTION

doi: 10.64898/2026.03.02.709032

Figure Lengend Snippet: (A-C) Huh7.5 cells were transduced with shCTRL, shATF6, shIRE1 or shPERK lentivirus to generate stable knockdowns of ATF6, IRE1 and PERK. (A) Successful silencing was confirmed by western blot analysis. (B-C) Cells were treated with DMSO or 0.1 µM Tg for 30 minutes and subsequently infected with HCVcc (MOI 0.2) for 72 hours. (B) Cells were fixed and stained for cell nuclei (DAPI; blue), lipid droplets (BODIPY-493; green) or HCV core (red) and imaged using a confocal microscope at 63x magnification. (C) Cellular supernatants were collected, and viral titer was assessed by focus-forming assay. (D) Huh7.5 cells were treated with DMSO or 1 µg/mL tunicamycin (Tm) for 4 hours. Cellular RNA was harvested and XBP1s and HERPUD1 mRNA expression was assessed by RT-qPCR. (E-G) Huh7.5 cells were pre-treated with DMSO or 1 µg/mL Tm for four hours, and subsequently infected with HCVcc (MOI 0.2) for 72 hours. (E) intracellular HCV RNA was assessed by RT-qPCR, (F) Cells were fixed and stained as described in (B) . (G) Viral titer was assessed by focus forming assay. Graphs shown mean +/− SEM from 3 independent experiments. RT-qPCR was performed in technical triplicate per biological replicate. Confocal microscopy data are representative of at least three biological replicates with at least three technical replicates per condition. *p<0.05, **p<0.01, ***p<0.0005, ****p<0.0001.

Article Snippet: Primary antibodies against ATF6 (Cell Signaling Technology (CST) 8089, dilution 1:1000), IRE1a (CST 3294, dilution 1:1000), PERK (CST 5683, dilution 1:1000), and beta-actin (Abcam ab8226, dilution 1:5000), and secondary antibodies Licor IRDye-680 goat anti-mouse and Licor IRDye-800 were used for western blot analysis.

Techniques: Transduction, Western Blot, Infection, Staining, Microscopy, Focus Forming Assay, Expressing, Quantitative RT-PCR, Confocal Microscopy

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Tumor Suppressor TPD52-Governed Endoplasmic Reticulum Stress is Modulated by APC Cdc20 .

doi: 10.1002/advs.202405441

Figure Lengend Snippet: Figure 1. TPD52 promotes ATF6 activation during the UPR. a) Anti-TPD52 immunoprecipitates (IPs) coupled with mass spectrometry analysis (MS) to identify TPD52-interacting proteins in T24 cells. IPs-MS results were subjected to enrichment analysis. b) The proteins were enriched in the “protein processing in endoplasmic reticulum” categories. c) Immunoblot (IB) analysis of whole cell lysates (WCL) and anti-TPD52 immunoprecipitates (IPs)

Article Snippet: Rabbit primary antibodies against ATF6 (65 880), eIF2α (5324), phospho-eIF2α (Ser 51) (3398), PERK (3192), IRE1α (3294), Bip (3177), cyclin D1 (2978), Flag-tag (14 793), HA-tag (3724), GSTtag (2625) and β-actin (4970) were all purchased from Cell Signaling Technology, Inc (Boston, MA, USA).

Techniques: Activation Assay, Mass Spectrometry, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Tumor Suppressor TPD52-Governed Endoplasmic Reticulum Stress is Modulated by APC Cdc20 .

doi: 10.1002/advs.202405441

Figure Lengend Snippet: Figure 2. TPD52 regulates UPR signals and ER size through ATF6. a) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from T24, 5637, and A375 cells stably expressing shTPD52 or Scr. Scr, Scramble. b) IB analysis of WCL derived from 253J, J82, and SW780 cells stably overexpressing TPD52 or EV. EV, empty vector. c) IB analysis of the WCL derived from wild-type (WT) or Tpd52-knockout (TPD52−/−) mice bladder tissues. d) Representative transmission electron micrographs (TEMs) image of T24 cells stably expressing shTPD52 or Scr. Where indicated, 1 μg mL−1 Tu was added before harvesting the cells. Scr, Scramble. Scale bars: 500 nm (white); 200 nm (red). e) ER thickness was quantified for the indicated TEM samples in (d). f) Spearman analysis of gene-expression data from patients with bladder cancer (BLCA, TCGA dataset, n = 411 samples) and skin cutaneous melanoma (SKCM, TCGA dataset, n = 472 samples) was used for depicting the correlation between TPD52 and unfolded protein response (UPR). g) IB analysis of WCL derived from 253J ATF6 knockdown or control cells transduced with TPD52 lentivirus or T24 TPD52 knockdown or control cells transduced with ATF6 lentivirus. TPD, TPD52. Scr, Scramble. EV, empty vector. h) Representative TEMs image of T24 ATF6 knockdown or control cells transduced with TPD52 lentivirus. Where indicated, 1 μg mL−1 Tu was added before harvesting the cells. Scr, Scramble. EV, empty vector. Scale bars: 500 nm (white); 200 nm (red). i. ER thickness was quantified for the indicated TEM samples in (h).

Article Snippet: Rabbit primary antibodies against ATF6 (65 880), eIF2α (5324), phospho-eIF2α (Ser 51) (3398), PERK (3192), IRE1α (3294), Bip (3177), cyclin D1 (2978), Flag-tag (14 793), HA-tag (3724), GSTtag (2625) and β-actin (4970) were all purchased from Cell Signaling Technology, Inc (Boston, MA, USA).

Techniques: Western Blot, Derivative Assay, Stable Transfection, Expressing, Plasmid Preparation, Knock-Out, Transmission Assay, Gene Expression, Knockdown, Control, Transduction

Journal: International journal of molecular sciences

Article Title: The Activation of Reticulophagy by ER Stress through the ATF4-MAP1LC3A-CCPG1 Pathway in Ovarian Granulosa Cells Is Linked to Apoptosis and Necroptosis.

doi: 10.3390/ijms24032749

Figure Lengend Snippet: Figure 3. Immunofluorescence of markers of the UPRer and autophagy in control and 5 µM tunicamycin-treated cells. (A): Colocalization of ATF4 (green) protein and ER tracker (red); (B): Colo- calization of CHOP (green) protein and ER tracker (red); (C): Colocalization of XBP1s (green) protein and ER tracker (red); (D): Colocalization of ATF6 (green) protein and ER tracker (red); (E): Colocal- ization of ATF4 (red) protein and GFP-LC3 (green); (F): Colocalization of CCPG1 (red) protein and GFP-LC3 (green); (G): Autophagic flux detection by mRFP-GFP-LC3B staining. At least 50 different cells from different fields were analyzed, and the dots were blindly counted by three different persons. The nuclei were stained with DAPI (blue). * p < 0.05.

Article Snippet: Primary antibodies against MAP1LC3A (ab52628, RRID: AB_881227), CHOP (ab11419, RRID: AB_298023), XBP1s (ab220783, RRID: AB_2920809), ATF4 (ab184909, RRID: AB_2819059), Gabarap (ab109364, RRID: AB_10861928), PCNA (ab29, RRID: AB_303394), calreticulin (ab92516, RRID: AB_10562796), pripk3 (ab187091, RRID: AB_2619685), mlkl (ab184718, RRID: AB_2755030), p-mlkl (ab195117, RRID: AB_2768156), STAT3 (ab32500, RRID: AB_2286741) and GRP78 (ab32618, RRID: AB_732737) were purchased from Abcam; primary antibodies against ATF6 (24169-1-AP, RRID: AB_2876891), β-actin (20536-1-AP, RRID: AB_10700003), CCPG1 (13861-1-AP, RRID: AB_2074010), and STAT1 (10144-2-AP, RRID: AB_2286875) were purchased from Proteintech; primary antibodies against cleaved-caspase-3 (9661, RRID: AB_2341188), and cleaved-caspase-8 (9496, RRID: AB_561381) were purchased from Cell Signalling; and a primary antibody against LC3B (L7543, RRID: AB_796155) was purchased from Sigma (Louis, MO, USA).

Techniques: Control, Staining

Journal: International journal of molecular sciences

Article Title: The Activation of Reticulophagy by ER Stress through the ATF4-MAP1LC3A-CCPG1 Pathway in Ovarian Granulosa Cells Is Linked to Apoptosis and Necroptosis.

doi: 10.3390/ijms24032749

Figure Lengend Snippet: Figure 5. Impaired ER proteostasis in CCPG1-knockdown cells. (A): Detection of the UPRer, reticu- lophagy and apoptotic marker molecules by Western blotting in CCPG1-knockdown granulosa cells; (B): Colocalization of ATF4 (green), CHOP (green), XBP1s (green), ATF6 (green), and ER tracker (red) in CCPG1-knockdown granulosa cells; (C): Detection of cellular apoptosis by flow cytometry in the control and CCPG1-knockdown cells; (D): Protein aggregate detection results. At least 50 different cells in different fields were analyzed, and the dots were blindly counted by three different individu- als. β-Actin was used as a reference protein, *** p < 0.001, ** p < 0.01, * p < 0.05. The nuclei are stained with DAPI (blue).

Article Snippet: Primary antibodies against MAP1LC3A (ab52628, RRID: AB_881227), CHOP (ab11419, RRID: AB_298023), XBP1s (ab220783, RRID: AB_2920809), ATF4 (ab184909, RRID: AB_2819059), Gabarap (ab109364, RRID: AB_10861928), PCNA (ab29, RRID: AB_303394), calreticulin (ab92516, RRID: AB_10562796), pripk3 (ab187091, RRID: AB_2619685), mlkl (ab184718, RRID: AB_2755030), p-mlkl (ab195117, RRID: AB_2768156), STAT3 (ab32500, RRID: AB_2286741) and GRP78 (ab32618, RRID: AB_732737) were purchased from Abcam; primary antibodies against ATF6 (24169-1-AP, RRID: AB_2876891), β-actin (20536-1-AP, RRID: AB_10700003), CCPG1 (13861-1-AP, RRID: AB_2074010), and STAT1 (10144-2-AP, RRID: AB_2286875) were purchased from Proteintech; primary antibodies against cleaved-caspase-3 (9661, RRID: AB_2341188), and cleaved-caspase-8 (9496, RRID: AB_561381) were purchased from Cell Signalling; and a primary antibody against LC3B (L7543, RRID: AB_796155) was purchased from Sigma (Louis, MO, USA).

Techniques: Knockdown, Marker, Western Blot, Cytometry, Control, Staining