Journal: Anesthesia & Analgesia

Article Title: Remifentanil Induces Cardio Protection Against Ischemia/Reperfusion Injury by Inhibiting Endoplasmic Reticulum Stress Through the Maintenance of Zinc Homeostasis

doi: 10.1213/ane.0000000000003414

Figure Lengend Snippet: Figure 2. Preconditioning with Rem rescues Zn2+ deficiency-induced ER stress in isolated ischemic reperfused rat hearts. Compared with the I/R group, Rem preconditioning reversed Zn2+ outflux from the heart (A) via inhibiting the expression of MTF1 (B and C) and its target gene ZnT1 (B and D). Rem attenuated ER stress by decreasing the expression of CHOP (E and F) and BIP (E and G), which were abolished by TPEN. *P < .05 versus sham; #P < .05 versus I/R; †P < .05 versus Rem + I/R. BIP indicates binding immunoglobulin protein; CHOP, CCAAT-enhancer-binding protein homolo- gous protein; ER, endoplasmic reticulum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; I/R, ischemia/reperfusion; MTF1, metal-responsive transcription factor 1; Rem, remifentanil; TPEN, N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine; ZnT1, zinc transporter protein 1; Zn2+, zinc.

Article Snippet: The membranes were blocked with 5% nonfat milk solution and incubated with primary antibodies, including antiMTF1 (1:1000; Biobyte, Claremont, CA), anti-ZnT1 (1:1000; Invitrogen, Carlsbad, CA), anti-CCAAT-enhancer-binding protein homologous protein (CHOP) (1:1000; Cell Signaling Technology [CST], Beverly, MA), anti-binding immunoglobulin protein (BIP) (1:1000; CST), anti-B-cell lymphoma 2 (Bcl2) (1:1000; CST), anti-Bcl2-associated X protein (Bax) (1:1000; Abcam, Cambridge, MA), anti-Caspase3 (1:1000; CST), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1500; CST) antibodies.

Techniques: Isolation, Expressing, Binding Assay

Journal: Anesthesia & Analgesia

Article Title: Remifentanil Induces Cardio Protection Against Ischemia/Reperfusion Injury by Inhibiting Endoplasmic Reticulum Stress Through the Maintenance of Zinc Homeostasis

doi: 10.1213/ane.0000000000003414

Figure Lengend Snippet: Figure 5. Effect of Rem on ZnT1 expression and ER stress in H9c2 cells overexpressed with MTF1. A, Confocal images of cells loaded with ER Tracker Red and Newport Green DCF. B, Western blotting images of ZnT1, CHOP, and BIP expressions. C–E, Summarized data for ZnT1, CHOP, and BIP expressions. *P < .05 versus control with vector; #P < .05 versus H/R with vector; †P < .05 versus H/R with vector; §P < .05 versus Rem + H/R with vector. Scale bar: 10 μm. BIP indicates binding immunoglobulin protein; CHOP, C/EBP homologous protein; DCF, dichlorodi- hydrofluoresceine; ER, endoplasmic reticulum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H/R, hypoxia/reoxygenation; H9c2, the embryonic rat heart–derived myogenic cell line H9c2; MTF1, metal-responsive transcription factor 1; Rem, remifentanil; Tag2B, pCMV-Tag2B; ZnT1, zinc transporter protein 1.

Article Snippet: The membranes were blocked with 5% nonfat milk solution and incubated with primary antibodies, including antiMTF1 (1:1000; Biobyte, Claremont, CA), anti-ZnT1 (1:1000; Invitrogen, Carlsbad, CA), anti-CCAAT-enhancer-binding protein homologous protein (CHOP) (1:1000; Cell Signaling Technology [CST], Beverly, MA), anti-binding immunoglobulin protein (BIP) (1:1000; CST), anti-B-cell lymphoma 2 (Bcl2) (1:1000; CST), anti-Bcl2-associated X protein (Bax) (1:1000; Abcam, Cambridge, MA), anti-Caspase3 (1:1000; CST), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1500; CST) antibodies.

Techniques: Expressing, Western Blot, Control, Plasmid Preparation, Binding Assay, Derivative Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: TMEM132A, a Novel Wnt Signaling Pathway Regulator Through Wntless (WLS) Interaction

doi: 10.3389/fcell.2020.599890

Figure Lengend Snippet: TMEM132A type I transmembrane protein localizes to the plasma membrane and ER and interacts with itself. (A) HeLa cells transiently expressing FL T132A-GFP, ER tranker of ER-RFP (upper panel) or plasma membrane tracker of PM-RFP (lower panel) were fixed, nuclei counterstained with Hoechst, and imaged under 100× oil objective lens. Fluorescent signals were pseudo-colored in green, red or gray as indicated. Scale bars: 5 μm. (B) Schematic representation of FL T132A-Flag consisting of S ignal peptide (SP), N -terminus, T ransmembrane (TM) domain and C -terminus (abbreviated as SNTC), truncated T132A-Flag of SNT or STC, and FL HA-tagged TMEM132A (T132A-HA). (C) Co-IP between FL T132A-Flag and T132A-HA in HEK 293T cells. (D) Co-IP of FL T132A-HA by FL and SNT, but not by STC of T132A-Flag in HEK 293T cells. * indicates non-specific bands.

Article Snippet: Antibodies for α-TMEM132A (Proteintech, #25301-1-AP), α-WLS (MilliporeSigma, #MABS87), α-GAPDH (MilliporeSigma, #G9545), α-Phospho-β-Catenin (Ser45) (CST, #9564), α-β-Catenin (CST, #8480), α-Flag (MilliporeSigma, #F1804), α-Myc (Thermo Fisher Scientific, #13-2500), α-HA (MilliporeSigma, #H6908) and α-turbo RFP (Origene, #TA150061) were used in Western blot.

Techniques: Clinical Proteomics, Membrane, Expressing, Co-Immunoprecipitation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: TMEM132A, a Novel Wnt Signaling Pathway Regulator Through Wntless (WLS) Interaction

doi: 10.3389/fcell.2020.599890

Figure Lengend Snippet: TMEM132A colocalizes with WLS. (A,B) HeLa cells co-transfected with FL T132A-Flag, FL WLS-HA, and ER-RFP were fixed and immunostained, nuclei counterstained with Hoechst. (C) HeLa cells co-transfected with FL T132A-GFP and FL WLS-RFP were fixed and nuclei counterstained with Hoechst. The fluorescence intensity in the boxed area was quantified along the indicated white arrow for both channels. Cells were imaged under 100× oil objective lens. Fluorescent signals were pseudo-colored in green, red, blue, or gray as indicated. Scale bars: 5 μm.

Article Snippet: Antibodies for α-TMEM132A (Proteintech, #25301-1-AP), α-WLS (MilliporeSigma, #MABS87), α-GAPDH (MilliporeSigma, #G9545), α-Phospho-β-Catenin (Ser45) (CST, #9564), α-β-Catenin (CST, #8480), α-Flag (MilliporeSigma, #F1804), α-Myc (Thermo Fisher Scientific, #13-2500), α-HA (MilliporeSigma, #H6908) and α-turbo RFP (Origene, #TA150061) were used in Western blot.

Techniques: Transfection, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: TMEM132A, a Novel Wnt Signaling Pathway Regulator Through Wntless (WLS) Interaction

doi: 10.3389/fcell.2020.599890

Figure Lengend Snippet: TMEM132A and WLS interact with each other. (A) Schematic representation of FL and truncated constructs of T132A-Flag and WLS-HA. (B) Co-IP of endogenous WLS by FL T132A-Flag (left), and co-IP of endogenous TMEM132A by FL WLS-HA (right) in HEK 293T cells. (C) Co-IP between FL T132A-Flag and FL WLS-HA in HEK 293T cells. (D) Co-IP of WLS-HA by FL, but not SN or C expression constructs lacking TM domain of T132A-Flag in HEK 293T cells. (E) Co-IP of WLS-HA by FL, SNT and STC of T132A-Flag in HEK 293T cells. (F) Co-IP of WLS-HA by FL and ST of T132A-Flag but not by the SNC construct lacking TM domain of T132A-Flag in HEK 293T cells. (G) Co-IP showing strong interaction of FL T132A-Flag with FL and truncation 1-264 of WLS-HA, and weak interaction with truncations 332–428 and 429–541 of WLS-HA in HEK 293T cells. * indicates non-specific band.

Article Snippet: Antibodies for α-TMEM132A (Proteintech, #25301-1-AP), α-WLS (MilliporeSigma, #MABS87), α-GAPDH (MilliporeSigma, #G9545), α-Phospho-β-Catenin (Ser45) (CST, #9564), α-β-Catenin (CST, #8480), α-Flag (MilliporeSigma, #F1804), α-Myc (Thermo Fisher Scientific, #13-2500), α-HA (MilliporeSigma, #H6908) and α-turbo RFP (Origene, #TA150061) were used in Western blot.

Techniques: Construct, Co-Immunoprecipitation Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: TMEM132A, a Novel Wnt Signaling Pathway Regulator Through Wntless (WLS) Interaction

doi: 10.3389/fcell.2020.599890

Figure Lengend Snippet: WNT3A and WNT5A protein levels are decreased upon loss of TMEM132A. (A) MEFs were isolated from crosses of Tmem132a +/– male and female mice to obtain WT, HET, and HOM embryonic fibroblasts, and TMEM132A protein levels were determined by Western blot. GAPDH served as an internal control. Relative band intensity is given below corresponding panel (normalized to GADPH). (B) MEFs were transfected with FL WNT3A-HA or WNT5A-HA, 48 hours later the culture medium was collected and the cells were lysed for Western blot with GAPDH as an internal control. Both intracellular and secreted levels of WNT3A or WNT5A were decreased by loss of TMEM132A, and band intensity was quantified (normalized to GADPH) in (C) . (D) TMEM132A specific siRNAs (#1 and #2) were used to knockdown TMEM132A compared to scramble siRNA (Ctrl) in HEK 293T cells and the efficiency was evaluated by real-time quantitative PCR 48 hours post transfection. GAPDH served as an internal control. (E) HEK 293T cells were co-transfected with scramble or TMEM132A specific siRNAs, and pCMV6-AC-RFP as a transfection control, and cell lysates collected 24 hours post transfection for Western blot. Relative band intensity is given below corresponding panel (normalized to GADPH). (F) HEK 293T cells were co-transfected with scramble or TMEM132A specific siRNAs, FL WNT3A-HA or WNT5A-HA, and FL WLS-Myc in indicated groups, and the culture medium and cell lysate were collected 48 hours post transfection for Western blot. Knockdown of TMEM132A results in decreased cytoplasmic and secreted WNT3A or WNT5A protein levels. Relative band intensity is given below corresponding panel (normalized to GADPH). * indicates non-specific band.

Article Snippet: Antibodies for α-TMEM132A (Proteintech, #25301-1-AP), α-WLS (MilliporeSigma, #MABS87), α-GAPDH (MilliporeSigma, #G9545), α-Phospho-β-Catenin (Ser45) (CST, #9564), α-β-Catenin (CST, #8480), α-Flag (MilliporeSigma, #F1804), α-Myc (Thermo Fisher Scientific, #13-2500), α-HA (MilliporeSigma, #H6908) and α-turbo RFP (Origene, #TA150061) were used in Western blot.

Techniques: Isolation, Western Blot, Control, Transfection, Knockdown, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cell and Developmental Biology

Article Title: TMEM132A, a Novel Wnt Signaling Pathway Regulator Through Wntless (WLS) Interaction

doi: 10.3389/fcell.2020.599890

Figure Lengend Snippet: TMEM132A positively regulates Wnt signaling pathway. (A) Schematic representation of co-culture and dual-luciferase assays. Topflash firefly luciferase reporter was used to measure canonical Wnt/β-Catenin signaling pathway activation in (B) , and pFR firefly luciferase reporter accompanied by pFA2-c-Jun were used to evaluate non-canonical Wnt/PCP signaling pathway activation in (C) . Constitutively expressed Renilla reniformis luciferase served as an internal control ( n = 3 biological replicates; p -value was calculated by Student’s t -test). (D) The levels of phospho-β-Catenin (Ser45) and total β-Catenin were determined by Western blot in whole cell lysate of untransfected wild type and TMEM132A mutant MEFs. (E) The ratio of phospho-β-Catenin (Ser45)/total β-Catenin was calculated from the band intensity in panel (D) . The result shows β-Catenin becomes unstable by loss of TMEM132A ( n = 3 biological replicates; p -value was calculated by Student’s t -test). (F) Real-time quantitative PCR was performed and shows relative transcription level of canonical Wnt/β-Catenin signaling pathway target genes Axin2 and Ccnd1 are downregulated by loss of TMEM132A ( n = 3 biological replicates; p -value was calculated by Student’s t -test). GAPDH served as an internal control.

Article Snippet: Antibodies for α-TMEM132A (Proteintech, #25301-1-AP), α-WLS (MilliporeSigma, #MABS87), α-GAPDH (MilliporeSigma, #G9545), α-Phospho-β-Catenin (Ser45) (CST, #9564), α-β-Catenin (CST, #8480), α-Flag (MilliporeSigma, #F1804), α-Myc (Thermo Fisher Scientific, #13-2500), α-HA (MilliporeSigma, #H6908) and α-turbo RFP (Origene, #TA150061) were used in Western blot.

Techniques: Co-Culture Assay, Luciferase, Activation Assay, Control, Western Blot, Mutagenesis, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cell and Developmental Biology

Article Title: TMEM132A, a Novel Wnt Signaling Pathway Regulator Through Wntless (WLS) Interaction

doi: 10.3389/fcell.2020.599890

Figure Lengend Snippet: TMEM132A stabilizes Wnt ligands and increases their binding with WLS. (A) Loss of TMEM132A destabilizes WNT protein levels. (Left panel) Scramble or TMEM132A siRNAs were transfected into HEK 293T cells followed by transfection with FL WNT3A or WNT5A-HA. After 24 hours the cells were treated with 100 μg/ml CHX and the protein levels of WNT3A or WNR5A were evaluated at the indicted time periods by Western blot. (Middle panel) TMEM132A knockdown efficiency before CHX treatment ( t = 0). GAPDH served as an internal control. (Right panel) Relative WNT3A (upper panel) or WNT5A protein (lower panel) remained after CHX treatment was calculated by measuring the Western blot band grayscale. (B) WNT glycosylation is not altered by TMEM132A depletion. HEK 293T cells were co-transfected with TMEM132A siRNAs and WNT3A or WNT5A-HA, and the glycosylation status of WNT3A or WNT5A was determined by Western blot. Tun at the indicated concentrations was used to block global glycosylation as a positive control. Relative band intensity is given below corresponding panel (normalized to GADPH). (C) WNT palmitoylation is not altered by TMEM132A depletion. HEK 293T cells co-transfected with scramble or TMEM132A specific siRNAs and FL WNT3A or WNT5A-HA were labeled with 100 μM palmitic acid alkyne (Alk-C16:0), lysed and subjected to IP and on-bead click chemistry reaction in the presence of biotin azide plus to determine WNT3A/5A palmitoylation level by Western blot. TMEM132A knockdown efficiency is shown, with GAPDH as an internal control. Relative band intensity is given below corresponding panel (normalized to GADPH except the α-Biotin panel which is normalized to α-HA signal). (D) Co-IP was performed to determine the interaction between WLS and WNT3A or WNT5A in the presence or absence of overexpressed TMEM132A in HEK 293T cells. The result shows enhanced WLS-WNT3A or WNT5A interaction by addition of TMEM132A. Relative band intensity is given below corresponding panel (normalized to α-Myc signal) (E) Co-IP of FL WNT3A-HA and WNT5A-HA by FL T132A-Flag in HEK 293T cells.

Article Snippet: Antibodies for α-TMEM132A (Proteintech, #25301-1-AP), α-WLS (MilliporeSigma, #MABS87), α-GAPDH (MilliporeSigma, #G9545), α-Phospho-β-Catenin (Ser45) (CST, #9564), α-β-Catenin (CST, #8480), α-Flag (MilliporeSigma, #F1804), α-Myc (Thermo Fisher Scientific, #13-2500), α-HA (MilliporeSigma, #H6908) and α-turbo RFP (Origene, #TA150061) were used in Western blot.

Techniques: Binding Assay, Transfection, Western Blot, Knockdown, Control, Glycoproteomics, Blocking Assay, Positive Control, Labeling, Co-Immunoprecipitation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: TMEM132A, a Novel Wnt Signaling Pathway Regulator Through Wntless (WLS) Interaction

doi: 10.3389/fcell.2020.599890

Figure Lengend Snippet: Schematic model of TMEM132A regulation of Wnt signaling pathway within signal sending cells. The TMEM132A homodimer (or oligomer) interacts with the Wnt ligand trafficking protein WLS to increase WNT binding and WNT stabilization, and to enhance WNT secretion and Wnt signaling pathway activation. In the absence of TMEM132A, Wnt ligand becomes unstable, and WLS–Wnt ligand interaction is weakened, leading to repressed Wnt signaling pathway.

Article Snippet: Antibodies for α-TMEM132A (Proteintech, #25301-1-AP), α-WLS (MilliporeSigma, #MABS87), α-GAPDH (MilliporeSigma, #G9545), α-Phospho-β-Catenin (Ser45) (CST, #9564), α-β-Catenin (CST, #8480), α-Flag (MilliporeSigma, #F1804), α-Myc (Thermo Fisher Scientific, #13-2500), α-HA (MilliporeSigma, #H6908) and α-turbo RFP (Origene, #TA150061) were used in Western blot.

Techniques: Binding Assay, Activation Assay

Journal: PLoS ONE

Article Title: Autoreactivity to Glucose Regulated Protein 78 Links Emphysema and Osteoporosis in Smokers

doi: 10.1371/journal.pone.0105066

Figure Lengend Snippet: A .) Anti-GRP78 autoantibody prevalence was not significantly associated with COPD severity (GOLD stages) . Numbers within columns denote subject n. B .) Anti-GRP78 autoantibodies are significantly associated with emphysema prevalence (%) in the smoking subjects. OR = odds ratio; CI = confidence interval. C .) Emphysema scores per CT were also significantly greater among those subjects with anti-GRP78 autoantibodies.

Article Snippet: Membranes were incubated with 1∶1000 dilutions of mouse anti-human GRP78 mAb (R&D Systems) at 4°C, followed by 1∶4000 dilutions of chicken anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques:

Journal: PLoS ONE

Article Title: Autoreactivity to Glucose Regulated Protein 78 Links Emphysema and Osteoporosis in Smokers

doi: 10.1371/journal.pone.0105066

Figure Lengend Snippet: A .) T scores (left panel) and osteoporosis prevalence (at either/both hip or spine) (right panel) among the smoking cohort. B .) Serum levels of bone turnover metabolite collagen type 1 cross-linked C-telopeptide (CTX) were greatest among smokers with anti-GRP78 autoantibodies. The lowest, second lowest, middle, second highest, and highest lines represent 10 th , 25 th , median, 75 th , and 90 th percentiles, respectively. Means are denoted by solid squares. C .) Serum levels of bone turnover metabolite type 1 (N-terminal) procollagen (P1NP) were greatest among smokers with anti-GRP78 autoantibodies. D .) The relationship between GRP78 autoantibody positivity and the concurrent co-existences of low BMD and emphysema in smokers is significant in both genders, but greatest in males.

Article Snippet: Membranes were incubated with 1∶1000 dilutions of mouse anti-human GRP78 mAb (R&D Systems) at 4°C, followed by 1∶4000 dilutions of chicken anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques:

Journal: PLoS ONE

Article Title: Autoreactivity to Glucose Regulated Protein 78 Links Emphysema and Osteoporosis in Smokers

doi: 10.1371/journal.pone.0105066

Figure Lengend Snippet: A .) Compared to normal lung (Left panel), emphysematous lungs (middle and right panels) demonstrated increased immunostaining in macrophages (yellow arrows) and alveolar epithelial cells (blue arrows). Magnification x100 left and middle panels, and magnification x400 in the right panel, n = 3. B .) GRP78 was also greater in bronchoalveolar lavage fluid (BALF) from emphysematous lungs compared to normal preparations. Lanes 1 and 8 are rGRP78 standards. Lanes 2–7 are BALF from individual emphysematous lung explants; lanes 9–14 are BALF from normal lung explants. All specimen lanes were loaded with equal amounts of BALF proteins. C .) Indirect immunofluorescent assays showed anti-GRP78 IgG isolated from patients bind to alveolar macrophages from normal lung explants (panel a), and osteoclasts derived from bone marrow (panel b). Normal human IgG control is illustrated in panel c.

Article Snippet: Membranes were incubated with 1∶1000 dilutions of mouse anti-human GRP78 mAb (R&D Systems) at 4°C, followed by 1∶4000 dilutions of chicken anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Immunostaining, Isolation, Derivative Assay, Control

Journal: PLoS ONE

Article Title: Autoreactivity to Glucose Regulated Protein 78 Links Emphysema and Osteoporosis in Smokers

doi: 10.1371/journal.pone.0105066

Figure Lengend Snippet: A .) Mean fluorescence intensity (MFI) for phosphorylated NFkB among paired, concurrent autologous CD14 + derived macrophages was increased in all 10 normal specimens after incubation with patient-derived autoantibodies to GRP78 (α-GRP78), relative to control cells treated with normal human IgG. Patient derived anti-GRP78 autoantibodies also increased macrophage production of IL-8 ( B .), CCL-2 ( C .) and MMP9 ( C .). Population means are denoted with a horizontal line.

Article Snippet: Membranes were incubated with 1∶1000 dilutions of mouse anti-human GRP78 mAb (R&D Systems) at 4°C, followed by 1∶4000 dilutions of chicken anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Fluorescence, Derivative Assay, Incubation, Control

Journal: PLoS ONE

Article Title: Autoreactivity to Glucose Regulated Protein 78 Links Emphysema and Osteoporosis in Smokers

doi: 10.1371/journal.pone.0105066

Figure Lengend Snippet: A .) Addition of GRP78 to PBMNC cultures increased CD4 T-cell proliferation (BrdU uptake) relative to media controls (no added protein) (n = 47), unlike addition of elastin split products (ESP). P c denotes alpha level corrected for multiple comparisons. B .) GRP78-induced CD4 T-cell proliferation was greatest among cultures from smokers with emphysema (n = 34). Specific indices (SI) of proliferation were calculated as %CD4 T-cells incorporating BrdU in GRP78-supplemented cultures minus incorporation in concurrent media controls. C .) GRP78 also increased percentages of CD4 T-cells that produced IFN-gamma again, unlike effects of ESP. D .) IFN-gamma production was also greatest in the CD4 T-cells from the emphysematous smokers. Specific indices (SI) were calculated as %CD4 T-cells producing IFN-gamma production in GRP78-supplemented cultures minus that of concurrent controls.

Article Snippet: Membranes were incubated with 1∶1000 dilutions of mouse anti-human GRP78 mAb (R&D Systems) at 4°C, followed by 1∶4000 dilutions of chicken anti-mouse IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Produced