Journal: Cell Biochemistry and Biophysics

Article Title: Interaction of L-SIGN with Hepatitis C Virus Envelope Protein E2 Up-Regulates Raf–MEK–ERK Pathway

doi: 10.1007/s12013-012-9505-4

Figure Lengend Snippet: a Expression of L-SIGN on NIH3T3 and NIH3T3/L-SIGN. Cells were incubated with mouse anti-L-SIGN mAb ( thick lines ) or isotype control ( dotted lines ). Expression of L-SIGN was detected by flow cytometry using FITC-conjugated goat anti-mouse IgG. The results are representative of three experiments. b Confocal microscopy was carried out to characterize cellular localization of L-SIGN on NIH3T3 and NIH3T3/L-SIGN. Cells were stained with mouse anti-L-SIGN mAb and FITC-conjugated goat anti-mouse IgG. The results are reproducible in two experiments, and representative fields are shown

Article Snippet: Horseradish peroxidase-conjugated goat anti-rabbit IgG and alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse IgG were from Vector Lab (Burlingame, CA, USA).

Techniques: Expressing, Incubation, Flow Cytometry, Confocal Microscopy, Staining

Journal: Cell Biochemistry and Biophysics

Article Title: Interaction of L-SIGN with Hepatitis C Virus Envelope Protein E2 Up-Regulates Raf–MEK–ERK Pathway

doi: 10.1007/s12013-012-9505-4

Figure Lengend Snippet: a Binding of HCV E2 protein to NIH3T3/L-SIGN. Cells were treated with E2 ( thick lines ) or left untreated ( dotted lines ) and the E2 binding was detected with mouse anti-E2 mAb and FITC-conjugated goat anti-mouse IgG by flow cytometry. b Inhibition of HCV E2 binding to NIH3T3/L-SIGN by antibodies against L-SIGN or DC-SIGN. Cells were incubated with mouse anti-L-SIGN mAb at a concentration of 4 μg/ml (II), 10 μg/ml (III) or 10 μg/ml anti-DC-SIGN mAb, and 10 μg/ml anti-L-SIGN mAb (IV) before the E2 incubation. The E2 binding was detected with goat anti-E2 Ab and FITC-conjugated rabbit anti-goat IgG in the presence or absence (I) of the antibody incubation. The percentage of marker-positive cells is indicated in each case. Data are representative of three experiments

Article Snippet: Horseradish peroxidase-conjugated goat anti-rabbit IgG and alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse IgG were from Vector Lab (Burlingame, CA, USA).

Techniques: Binding Assay, Flow Cytometry, Inhibition, Incubation, Concentration Assay, Marker

Journal: Respiratory Research

Article Title: RAGE inhibition alleviates lipopolysaccharides-induced lung injury via directly suppressing autophagic apoptosis of type II alveolar epithelial cells

doi: 10.1186/s12931-023-02332-6

Figure Lengend Snippet: LPS-induced autophagic death in A549 cells depended on RAGE activation. A Treated A549 cells with siRAGE and detected the RAGE mRNA expression. The expression of Ager genes was decreased by 50%. B Detected the immunoblots of RAGE as well as Beclin1, LC3 II/I and cleaved Caspase 3 in A549 cells, each experiment was repeated more than three times. C The cell survival rate of A549 cells in response to siRAGE pre-treatment followed by LPS stimulation. Data were obtained from CCK-8 experiments and performed more than three times. D Immunofluorescence of cleaved Caspase3 to detect the apoptosis of A549 cells, spinning disk confocal microscopy at × 40 magnification (scale bar 50 μm). E Detected the immunoblots of STAT3 and phosphorylation STAT3 (p-STAT3) in A549 cells, each experiment was repeated more than three times. *Indicates the significant difference compared with the control group. # Indicates the significant difference compared with LPS group. P < 0.05, differences in characteristics between groups were analyzed using the Kruskal–Wallis test with Dunn’s post hoc tests

Article Snippet: Anti-RAGE (1:1000, #6996S; Cell Signaling Technology, Inc., MA, USA), anti-STAT3 (1:1000, #12640; Cell Signaling Technology, Inc., MA, USA), anti-phosphorylated STAT3 (1:2000, #9145; Cell Signaling Technology, Inc., MA, USA), anti-cleaved caspase 3 (1:1000, #9664; Cell Signaling Technology, Inc., MA, USA), anti-LC3II/I (1:1000, #4108; Cell Signaling Technology, Inc., MA, USA), anti-Beclin1 (1:1000, #3495; Cell Signaling Technology, Inc., MA, USA) and anti-GADPH (1:1000, #2118; Cell Signaling Technology, Inc., MA, USA) were used as primary antibodies.

Techniques: Activation Assay, Expressing, Western Blot, CCK-8 Assay, Immunofluorescence, Confocal Microscopy, Phospho-proteomics, Control

Journal: International Journal of Biological Sciences

Article Title: Metformin Alleviates Liver Metabolic Dysfunction in Polycystic Ovary Syndrome by Activating the Ethe1/Keap1/PINK1 Pathway

doi: 10.7150/ijbs.104778

Figure Lengend Snippet: Metformin improves mitochondrial function in the liver of PAMH-induced PCOS mice. Malondialdehyde (MDA) (A) , superoxide dismutase (SOD) (B) , glutathione (GSH) (C) , GSH/GSSG (D) , ATP (E) , and mitochondrial DNA (mtDNA) (F) in liver tissues from the control, PAMH-induced, and metformin treatment groups. (G, H) Reactive oxygen species levels in liver tissues labeled with dihydroethidium (DHE) as observed under a fluorescence microscope (G) . Mean fluorescence intensity of DHE in each group (H) . (I-M) Mitochondrial respiratory chain complexes: Complex I (NADH dehydrogenase) (I) , Complex II (succinate-coenzyme Q reductase) (J) , Complex III (ubiquinol-cytochrome c oxidoreductase) (K) , Complex IV (cytochrome C oxidase) (L) , and Complex V (ATP synthase) (M) . (N-Q, S, V) Markers related to mitochondrial autophagy (Mfn2, DRP1, LC3, P62, TOMM20, VDAC1, Nrf2, Keap1, PINK1, and Parkin) in total protein extraction from liver tissue based on western blotting (N, P) . Protein relative expression levels were calculated in the control group (orange bars), PAMH group (pink bars), and PAMH+Met group (purple bars) and also shown (O, Q, S, V) . (R, T, U) Expression of p62 and LC3 in each group. All error bars are mean values ± SD, p- values were determined by unpaired two-tailed Student's t test ( n = 3) in independent biological experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The PVDF membranes were incubated with anti-Ethe1 (1:2000, 27786-1-AP, Proteintech), anti-ETFDH polyclonal (1:2000, 11109-1-AP, Proteintech), NRF2,NFE2L2 polyclonal antibody (1:5000, 16396-1-AP, Proteintech), anti-Keap1 (1:5000, 10503-2-AP, Proteintech), PARK2/Parkin polyclonal antibody (1:2000, 14060-1-AP, Proteintech), PINK1 polyclonal antibody (1:500, 23274-1-AP, Proteintech), MFN2 polyclonal antibody (1:5000, 12186-1-AP, Proteintech), DRP1 (C-terminal) polyclonal antibody (1:2000, 12957-1-AP, Proteintech), anti-TOMM20 antibody (1:2000, ab186735, Abcam), VDAC1/Porin polyclonal antibody (1:2000, 55259-1-AP, Proteintech), P62, SQSTM1 polyclonal antibody (1:10000, 18420-1-AP, Proteintech), anti-LC3B antibody (1:2000, ab192890, Abcam), anti-βactin (1:5000; AC026, Abclonal), COXIV polyclonal antibody (1:5000, 11242-1-AP, Proteintech), and Histone H3 polyclonal antibody (1:5000, 17168-1-AP, Proteintech) at 25 °C for 2 h. After incubation with the corresponding secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit (1:5000, ZB2301, Zsbio) and horseradish peroxidase-conjugated goat anti-mouse (1:5000, ZB2305, Zsbio)) for 1 h at 20-25 °C, the blots were visualized using SuperSignal-enhanced chemiluminescent substrate solution (WBULS0100, Millipore).

Techniques: Control, Labeling, Fluorescence, Microscopy, Protein Extraction, Western Blot, Expressing, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Metformin Alleviates Liver Metabolic Dysfunction in Polycystic Ovary Syndrome by Activating the Ethe1/Keap1/PINK1 Pathway

doi: 10.7150/ijbs.104778

Figure Lengend Snippet: Metformin improves mitophagy in damaged hepatocytes. (A, B) Intracellular ATP levels of control (orange bars), DHEA- and FFA-induced (pink bars), and Met-treated (purple bars) cells. (C, D, G, H) Expression of Ethe1 and key molecules related to mitochondrial autophagy (Mfn2, DRP1, PINK1, Parkin, and TOMM20) in each group (colors as described for A and B ). (F, F) MtDNA copy number for each group. (I, K, L) Co-localization of mitochondria with LC3 and Mito-Tracker Red, PINK1, and Mito-Tracker Red under confocal microscopy in the control, DHEA- and FFA-induced, and Met-added cells. (I) Mander's co-localization coefficients (K, L) . (J) Mean fluorescence intensity of JC-1 in each group. All error bars are mean values ± SD, p- values were determined by unpaired two-tailed Student's t test ( n = 3) in independent biological experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The PVDF membranes were incubated with anti-Ethe1 (1:2000, 27786-1-AP, Proteintech), anti-ETFDH polyclonal (1:2000, 11109-1-AP, Proteintech), NRF2,NFE2L2 polyclonal antibody (1:5000, 16396-1-AP, Proteintech), anti-Keap1 (1:5000, 10503-2-AP, Proteintech), PARK2/Parkin polyclonal antibody (1:2000, 14060-1-AP, Proteintech), PINK1 polyclonal antibody (1:500, 23274-1-AP, Proteintech), MFN2 polyclonal antibody (1:5000, 12186-1-AP, Proteintech), DRP1 (C-terminal) polyclonal antibody (1:2000, 12957-1-AP, Proteintech), anti-TOMM20 antibody (1:2000, ab186735, Abcam), VDAC1/Porin polyclonal antibody (1:2000, 55259-1-AP, Proteintech), P62, SQSTM1 polyclonal antibody (1:10000, 18420-1-AP, Proteintech), anti-LC3B antibody (1:2000, ab192890, Abcam), anti-βactin (1:5000; AC026, Abclonal), COXIV polyclonal antibody (1:5000, 11242-1-AP, Proteintech), and Histone H3 polyclonal antibody (1:5000, 17168-1-AP, Proteintech) at 25 °C for 2 h. After incubation with the corresponding secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit (1:5000, ZB2301, Zsbio) and horseradish peroxidase-conjugated goat anti-mouse (1:5000, ZB2305, Zsbio)) for 1 h at 20-25 °C, the blots were visualized using SuperSignal-enhanced chemiluminescent substrate solution (WBULS0100, Millipore).

Techniques: Control, Expressing, Confocal Microscopy, Fluorescence, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Metformin Alleviates Liver Metabolic Dysfunction in Polycystic Ovary Syndrome by Activating the Ethe1/Keap1/PINK1 Pathway

doi: 10.7150/ijbs.104778

Figure Lengend Snippet: Metformin alleviates FFA- and DHEA-induced hepatocyte injury by regulating the Ethe1/Keap1/PINK1 pathway through ETFDH. (A, B) Expression of Keap1 in cells and Nrf2 in the nucleus and cytoplasm of control, DHEA-induced, and Met-treated cells (A) . Gray value levels (B) . (C-D) Expression of Keap1 in cells and Nrf2 in the nucleus and cytoplasm from the DHEA-induced, si-Ethe1, and Met-treated cells (C) . Relative gray value levels (D) . (F) Experimental design flowchart for Nrf2 and PINK1 functions. (E, G, H) CO-IP experiments verified the interaction between Ethe1 and ETFDH as well as the interaction between Ethe1 and Keap1 (E) in control group (orange bars), DHEA group (pink bars), and DHEA+Met group (purple group) (G, H) . (I, J) TG concentrations (I) . Representative Oil Red O staining imagines for each group (J) . (K, L-P) Each group of cells was labeled with Mitosox TM Red, DCFH-DA, and JC-1 probes, and the co-localization of LC3 and PINK1 with Mito-Tracker Red in cells from different groups was observed under a confocal microscope (K) . Mean fluorescence intensity of Mitosox TM Red, DCFH-DA, and JC-1 (L-N) . Mander's co-localization coefficients (O, P) . (Q-T) Expression of molecules related to mitochondrial autophagy (LC3, DRP1, PINK1, Parkin, and TOMM20) (Q) in the DHEA/FFAs+Met+si NC group (orange bars), DHEA/FFAs+Met+si Nrf2 group (pink bars), and DHEA/FFAs+Met+si PINK1 group (purple bars) (R-T) . All error bars are mean values ± SD, p- values were determined by unpaired two-tailed Student's t test ( n = 3) in independent biological experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The PVDF membranes were incubated with anti-Ethe1 (1:2000, 27786-1-AP, Proteintech), anti-ETFDH polyclonal (1:2000, 11109-1-AP, Proteintech), NRF2,NFE2L2 polyclonal antibody (1:5000, 16396-1-AP, Proteintech), anti-Keap1 (1:5000, 10503-2-AP, Proteintech), PARK2/Parkin polyclonal antibody (1:2000, 14060-1-AP, Proteintech), PINK1 polyclonal antibody (1:500, 23274-1-AP, Proteintech), MFN2 polyclonal antibody (1:5000, 12186-1-AP, Proteintech), DRP1 (C-terminal) polyclonal antibody (1:2000, 12957-1-AP, Proteintech), anti-TOMM20 antibody (1:2000, ab186735, Abcam), VDAC1/Porin polyclonal antibody (1:2000, 55259-1-AP, Proteintech), P62, SQSTM1 polyclonal antibody (1:10000, 18420-1-AP, Proteintech), anti-LC3B antibody (1:2000, ab192890, Abcam), anti-βactin (1:5000; AC026, Abclonal), COXIV polyclonal antibody (1:5000, 11242-1-AP, Proteintech), and Histone H3 polyclonal antibody (1:5000, 17168-1-AP, Proteintech) at 25 °C for 2 h. After incubation with the corresponding secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit (1:5000, ZB2301, Zsbio) and horseradish peroxidase-conjugated goat anti-mouse (1:5000, ZB2305, Zsbio)) for 1 h at 20-25 °C, the blots were visualized using SuperSignal-enhanced chemiluminescent substrate solution (WBULS0100, Millipore).

Techniques: Expressing, Control, Co-Immunoprecipitation Assay, Staining, Labeling, Microscopy, Fluorescence, Two Tailed Test

Journal: Iranian Journal of Microbiology

Article Title: Yeast-mediated display: probing Helicobacter pylori HopQ and CEACAM1 interaction

doi: 10.18502/ijm.v17i6.20366

Figure Lengend Snippet: SDS-PAGE and Western blot analyses. In panel A, the left image shows Coomassie Blue staining of S. cerevisiae expressing Aga2-C1ND (lanes 1 and 2). The middle blot shows two distinct bands at 50 kDa and 24 kDa detected using an HRP-conjugated anti-GFP antibody. Lane 1 corresponds to S. cerevisiae expressing the GFP-C1ND fusion construct. The presence of a 27 kDa band suggests cleavage of EGFP from its fusion partner. The right image shows signals detected by the anti-C1ND antibody for non-EGFP-tagged C1ND. The 24 kDa band corresponds to the Aga2-C1ND fusion protein. In panel B, the left image shows positive signals detected using the anti-C1ND antibody. Lane 1 shows the expression of the GFP-C1ND fusion protein, with bands at 24 kDa and 50 kDa. Lanes 2 and 3 represent strains expressing the Aga2-C1ND fusion protein, showing a band at 24 kDa. The positive control (lane 4) expresses the intact C1ND fragment, displaying a band at 12 kDa. The right image shows detection of the EGFP-C1ND fusion protein (50 kDa, lane 1) in a positive transformant using an HRP-conjugated anti-His-tag antibody. The positive control in this blot (lane 2) corresponds to an irrelevant His-tagged protein. In all blots, “C−“ indicates non-transformed lysate. Blots in panels A and B were developed using ECL and DAB staining, respectively.

Article Snippet: Immunoblots were probed overnight at 4°C with HRP-labeled antibodies against GFP (1:2000; Acris, USA), His-tag (1:1000; Sigma, USA), and C1ND (1:3000; Sigma, USA).

Techniques: SDS Page, Western Blot, Staining, Expressing, Construct, Positive Control, Transformation Assay

Journal: Iranian Journal of Microbiology

Article Title: Yeast-mediated display: probing Helicobacter pylori HopQ and CEACAM1 interaction

doi: 10.18502/ijm.v17i6.20366

Figure Lengend Snippet: Confocal microscopy images showing the surface expression of the Aga2-EGFP-G4S-C1ND fusion protein in S. cerevisiae , indicated by arrows. (A) Green fluorescence from the EGFP tag. (B) Red surface fluorescence of recombinant yeast cells stained with PE-conjugated anti-GFP antibody. (C) Merged image of panels A and B. (D) Brightfield image. (E) No fluorescence in the negative control. Scale bar: 5 μm

Article Snippet: Immunoblots were probed overnight at 4°C with HRP-labeled antibodies against GFP (1:2000; Acris, USA), His-tag (1:1000; Sigma, USA), and C1ND (1:3000; Sigma, USA).

Techniques: Confocal Microscopy, Expressing, Fluorescence, Recombinant, Staining, Negative Control

Journal: bioRxiv

Article Title: Adenine Nucleotide Translocase regulates the airway epithelium, mitochondrial metabolism and ciliary function

doi: 10.1101/2020.05.18.101378

Figure Lengend Snippet: A) Normal human airway tissue was stained for ANT1 (ab102032) or ANT2/3 (ab222843) and imaged by confocal microscopy. ANT (shown in red) localizes to the mitochondria (arrowhead) and to the ciliary layer (full arrow). The white inset shows additional detail. Representative of n=3-4 different subjects. B) ANT1 (magenta, stained in 555 channel) colocalization with TOM20 (green, stained in 647 channel) noted by an arrowhead, imaged by confocal microscopy. Scale bar, 10 μm. C) ANT1 (magenta, stained in 555 channel) colocalization with acetylated tubulin (green, stained in 647 channel) at the plasma membrane (arrowhead) and ciliary layer (full arrow), imaged by confocal microscopy. Scale bar, 10 μm. D) Human lung tissue from control and COPD patients stained for ANT1 (magenta, 555 channel) and acetylated tubulin (647 channel), imaged by confocal microscopy. Representative of n=3-5 subject per group. E) NHBEs grown at air liquid interface stained for native ANT1 or ANT2/3, demonstrating localization of ANT to the mitochondrial layer (arrowhead) and ciliary layer (full arrow). The white inset shows additional detail. F) ANT2/3 localizes to the plasma membrane in NHBE cells. NHBE cells were stained for ANT2/3 (without prior permeabilization) followed by secondary antibody Alexa 555 (magenta), then permeabilized and repeat staining with anti-ANT2/3 followed by a secondary antibody Alexa 488 (green). The white is enlarged on the right. Scale bar, 10 μm. n=3 inserts. Images of cells where no permeabilization was used may be found in supplementary Fig. S4E.

Article Snippet: Human and mouse lung sections were stained for colocalization with primary and secondary antibodies: ANT1 (Abcam #ab102032 rabbit polyclonal, 1:1000), pan ANT (anti-ANT1/2/3 Abcam, #ab110322, mouse monoclonal 1:100), ANT2 (5H7, from S. Claypool, 1:100), ANT2 (Abcam, #ab118076, mouse monoclonal, 1:100), ANT2 (Abcam, #ab222843, 1:1000), ANT2/3 (Abcam, #ab230545, 1:100), acetylated α-tubulin (Cell Signaling, #5335T Rabbit at 1:2000 and Invitrogen, #32-2700 Mouse at 1:2000), TOM20 (Abcam, #ab56783 and Cell signaling, #42406S), NPHP4 (Atlas Antibodies, #HPA065526), GFP (Aves Labs Inc, 1:1000), goat anti-mouse, anti-rabbit or anti-chicken Alexa 488, 555 and 647 (Molecular Probes).

Techniques: Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: Adenine Nucleotide Translocase regulates the airway epithelium, mitochondrial metabolism and ciliary function

doi: 10.1101/2020.05.18.101378

Figure Lengend Snippet: A) Lentiviral overexpression of ANT2-GFP and GFP control vector in differentiated NHBE cells. Cells were stained with anti-GFP (green, Alexa 488) and anti-acetylated tubulin (magenta, Alexa 647) and imaged with confocal microscopy. Ciliated cells are noted with an arrowhead. Scale bar, 5 μm. Grayscale insets are included for each color channel. The composite color inset is enlarged on the right. B) Normal mouse airway tissue was stained for ANT1 (ab102032) or ANT2/3 (ab222843) and imaged by confocal microscopy. ANT (shown in red) localizes to the mitochondria and to the plasma membrane/ciliary layer (full arrow). The white inset shows additional detail, scale bar 5 μm. Representative of n=3. C) Mouse lungs (air versus smoke-exposed for 6 months) stained for ANT1 (magenta, ab102032), acetylated α-tubulin (green) and DAPI (blue). n=5 mice per group. Scale bar, 5 μm. D) Staining of wildtype C57BL/6EiJ and ant1 null mouse airways for ANT1 (ab102032). Plasma membrane localization is noted by the arrowhead and ciliary layers by the full arrow. Scale bar, 5 μm. E) ANT1 (magenta) and acetylated α-tubulin (green) in mouse upper respiratory and olfactory epithelium, scale bar 10 μm. Full arrows note apical plasma membrane ANT while arrowheads note mitochondrial ANT. Apical membrane ANT is present in motile ciliated respiratory epithelium compared to non-motile ciliated olfactory epithelium. The white inset is enlarged in the panel, scale bar 3 μm.

Article Snippet: Human and mouse lung sections were stained for colocalization with primary and secondary antibodies: ANT1 (Abcam #ab102032 rabbit polyclonal, 1:1000), pan ANT (anti-ANT1/2/3 Abcam, #ab110322, mouse monoclonal 1:100), ANT2 (5H7, from S. Claypool, 1:100), ANT2 (Abcam, #ab118076, mouse monoclonal, 1:100), ANT2 (Abcam, #ab222843, 1:1000), ANT2/3 (Abcam, #ab230545, 1:100), acetylated α-tubulin (Cell Signaling, #5335T Rabbit at 1:2000 and Invitrogen, #32-2700 Mouse at 1:2000), TOM20 (Abcam, #ab56783 and Cell signaling, #42406S), NPHP4 (Atlas Antibodies, #HPA065526), GFP (Aves Labs Inc, 1:1000), goat anti-mouse, anti-rabbit or anti-chicken Alexa 488, 555 and 647 (Molecular Probes).

Techniques: Over Expression, Plasmid Preparation, Staining, Confocal Microscopy