Journal: Genes & development

Article Title: TCF3 alternative splicing controlled by hnRNP H/F regulates E-cadherin expression and hESC pluripotency.

doi: 10.1101/gad.316984.118

Figure Lengend Snippet: Figure 3. hnRNP H/F proteins bind exonic regulatory elements in exon 18b. (A, left) Diagram of wild-type and SWAP constructs in which the positions of exon 18a and exon 18b are switched. (Right) 32P RT–PCR assays of RNAs extracted from transfected HeLa cells using T7 primer and Ex19R. PCR products were digested with PstI. (B, left) Diagram of point-swapping mutant constructs. The details of a series of mutants are described in Supplemental Figure S4. (Right) 32P RT–PCR assays of RNAs extracted from transfected HeLa cells using T7 primer and Ex19R. PCR products were digested with PstI or HaeII. The E12 (exon18a inclusion) percentage was calculated by dividing exon 18a signal by the sum of exon 18a and 18b signals and is indicated below. (C) 32P RT–PCR assays of additional point-swapping mu- tants in sections 3 and 6 in HeLa cells. (D, top) Sequence comparison of sections 3-2 and 6-2. The putative hnRNP H/F-binding sites are underlined. The RNA affinity assay used biotinylated 3-2 and 6-2 short RNA oligonucleotides. Isolated proteins were analyzed by Western blot using the indicated antibodies.

Article Snippet: Western blotting Cells were lysed with SDS sample buffer (50 mM Tris at pH 7.4, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxy- cholate, 1× proteinase inhibitor cocktail [Biotools]), resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary antibodies detecting CDH1 (1:1000; Cell Signaling), hnRNP H1 (1:1000; Bethyl Laboratories), hnRNP F (1:1500; Santa Cruz Biotechnology), GAPDH (1:20,000; Sigma), or ACTB (1:30,000; Sigma) overnight at 4°C.

Techniques: Construct, Reverse Transcription Polymerase Chain Reaction, Transfection, Mutagenesis, Sequencing, Comparison, Binding Assay, Isolation, Western Blot

Journal: Genes & development

Article Title: TCF3 alternative splicing controlled by hnRNP H/F regulates E-cadherin expression and hESC pluripotency.

doi: 10.1101/gad.316984.118

Figure Lengend Snippet: Figure 4. hnRNP H/F expression and recruitment to exonic regulatory elements decrease during differentiation. (A) E12/E47 ratio was examined by 32P RT–PCR. PCR products were digested by PstI. (B) Expression of pluripotency and differentiation markers as determined by quantitative PCR (qPCR) after 8 d of differentiation. The P-value was determined by two-tailed Student’s t-test. (∗) P < 0.05. (C) CLIP analysis to measure the hnRNP H protein enrichment on exon 18b of TCF3 mRNA by qPCR after 8 d of differentiation. Data were nor- malized to hnRNP H immunoprecipitation signal in undifferentiated H9 cells. All results represent the means ± SD from at least three independent experiments. (∗) P < 0.05. (D) Expression of hnRNP H and hnRNP F during 8 d of differentiation of H9 cells. mRNA levels were determined by qPCR. (E) Protein levels were analyzed by Western blot using the indicated antibodies (left), and the bar graph shows its quantification (right).

Article Snippet: Western blotting Cells were lysed with SDS sample buffer (50 mM Tris at pH 7.4, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxy- cholate, 1× proteinase inhibitor cocktail [Biotools]), resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary antibodies detecting CDH1 (1:1000; Cell Signaling), hnRNP H1 (1:1000; Bethyl Laboratories), hnRNP F (1:1500; Santa Cruz Biotechnology), GAPDH (1:20,000; Sigma), or ACTB (1:30,000; Sigma) overnight at 4°C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Two Tailed Test, Protein Enrichment, Immunoprecipitation, Western Blot

Journal: Genes & development

Article Title: TCF3 alternative splicing controlled by hnRNP H/F regulates E-cadherin expression and hESC pluripotency.

doi: 10.1101/gad.316984.118

Figure Lengend Snippet: Figure 5. hnRNP H1 and hnRNP F regulate TCF3 splicing and hESC morphology. (A) E12/E47 ratio in hESCs after the indicated siRNA treatment analyzed by 32P RT–PCR. RT–PCR products were digested by PstI and resolved by 5% PAGE. (Top) The E12 percentage is in- dicated below. (Bottom) Immunoblots showing protein levels after the indicated siRNA treatment. (B) E12/E47 ratio in 293T cells after the indicated siRNA treatment analyzed by 32P RT–PCR. (C) Splicing assays analyzed by 32P RT–PCR using T7 primer and Ex19R. The TCF3 minigene and the indicated expression plasmids were transfected into 293T cells. Total RNA from the transfected cells was used for 32P RT–PCR. (D) Cell morphology of H9 hESCs transfected by the indicated siRNAs. (E) Expression of pluripotency markers in hnRNP H/F knockdown cells, determined by qPCR. (F) Expression of differentiation markers in H9 hESCs after the indicated siRNA treatment, determined by qPCR.

Article Snippet: Western blotting Cells were lysed with SDS sample buffer (50 mM Tris at pH 7.4, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxy- cholate, 1× proteinase inhibitor cocktail [Biotools]), resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary antibodies detecting CDH1 (1:1000; Cell Signaling), hnRNP H1 (1:1000; Bethyl Laboratories), hnRNP F (1:1500; Santa Cruz Biotechnology), GAPDH (1:20,000; Sigma), or ACTB (1:30,000; Sigma) overnight at 4°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Knockdown

Journal: eLife

Article Title: mTORC1-induced retinal progenitor cell overproliferation leads to accelerated mitotic aging and degeneration of descendent Müller glia

doi: 10.7554/elife.70079

Figure Lengend Snippet: Figure 6. Rescue of Tsc1fl/fl;Tyrp1-Cre mouse retinal phenotypes by concomitant deletion of hypoxia-induced factor 1-alpha (Hif1a). (A) Levels of senescence markers in P30 mouse retinas with indicated genotypes were analyzed by Western blot (WB). Relative amounts of proteins used in each sample were determined by WB detection of β-actin. (B) Distributions of mTORC1-active cells, which are positive to pS6, MG, which are positive to Sox9, and senescent cells, which are positive to β-gal and p53, were examined by immunostaining of the eye sections. Expression of those markers in the Cre-affected cells were determined by comparing the expression of the Cre reporter R26tdTom. (C) Numbers of Sox9-positive MG in the mouse retinas were counted and the relative numbers are shown in the graph. (D) R26tdTom-negative wild-type cells and R26tdTom-positive Cre-affected cells in the retinas were determined byFACS and shown in the graph. (E) The lengths of telomeres of the cells isolated from P30 mouse retinas with the indicated genotypes were compared with that of control sample, and their relative values are shown in the graph. Error bars denote SD. Numbers of samples analyzed are shown in the graphs. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: 70079 18 of 27 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- BrdU(CldU) (Rat monoclonal) Novus Biologicals NB500- 169 IHC (1:200) Antibody Anti- BrdU(IdU) (Mouse monoclonal) EXBIO 11–286 C100 IHC (1:200) Antibody Anti- Brn3b (Rabbit polyclonal) Santa Cruz Biotechnology sc- 31989 IHC (1:200) Antibody Anti- Calbindin (Rabbit polyclonal) Swant Inc. CB- 38 IHC (1:200) Antibody Anti- Cdo (Goat polyclonal) R&D Systems AF2429 IHC (1:200) Antibody Anti- c- myc (Mouse monoclonal) Santa Cruz Biotechnology sc40 WB (1:1000) Antibody Anti- Cleaved caspase- 3 (Rabbit polyclonal) Cell Signaling Technology 9661 IHC (1:200) Antibody Anti- Ezrin (Mouse monoclonal) Invitrogen Biotechnology 35–7300 IHC (1:200) Antibody Anti- GFAP (Rabbit polyclonal) Abcam ab48050 IHC (1:200) Antibody Anti- GFP (Chicken polyclonal) Abcam ab13970 IHC (1:2000) Antibody Anti- Glutamine synthase (Rabbit polyclonal) Sigma- Aldrich G- 2781 IHC (1:200) Antibody Anti- Hif1a (Mouse monoclonal) R&D Systems MAB1536 WB (1:1000) Antibody Anti- Hif2α/Epas1 (Rabbit polyclonal) Novus Biologicals NB100- 122 WB (1:1000) Antibody Anti- Hk2 (Rabbit polyclonal) Cell Signaling Technology 2867 WB 1:1,000 IHC (1:200) Antibody Anti- Hydroxy- Hif1alpha (Rabbit polyclonal) Cell Signaling Technology 3434 WB (1:1000) Antibody Anti- M- opsin Merck Millipore AB5405 IHC (1:200) Antibody Anti- Otx2 (Rabbit polyclonal) Abcam ab25985 IHC (1:200) Antibody Anti- Otx2 (Rabbit polyclonal) Abcam ab183951 IHC (1:200) Antibody Anti- Otx2 (Goat polyclonal) R&D Systems AF1979- SP IHC (1:200) Antibody Anti- p21/Cip1 (Mouse monoclonal) Santa Cruz Biotechnology SC817 WB (1:1000) IHC (1:200) Antibody Anti- p53 (Mouse monoclonal) Merck Millipore OP03- 100 WB (1:1000) IHC (1:200) Antibody Anti- Pax6 (Rabbit polyclonal) Covance PRB- 278P IHC (1:200) Antibody Anti- Phospho Smad1/5(S463/465) (Rabbit polyclonal) Invitrogen Biotechnology 700047 IHC (1:200) Antibody Anti- phospho Smad2(ser465/467) (Rabbit polyclonal) - Cell Signaling Technology 18338 IHC (1:200) Antibody Anti- phospho- Histone H3(S10) (pH3; Rabbit polyclonal) Merck Millipore 04–1093 IHC (1:200) Antibody Anti- phospho- S6(S235/236) (pS6; Rabbit polyclonal) Cell Signaling Technology 2211 WB (1:1000) IHC (1:200) Continued Continued on next page Lim et al. eLife 2021;10:e70079.

Techniques: Western Blot, Immunostaining, Expressing, Isolation, Control

Journal: eLife

Article Title: mTORC1-induced retinal progenitor cell overproliferation leads to accelerated mitotic aging and degeneration of descendent Müller glia

doi: 10.7554/elife.70079

Figure Lengend Snippet: Figure 7. Hif1a supports mTORC1-induced retinal progenitor cell (RPC) proliferation through the expression of glycolytic enzymes. (A) The Cre-affected cells in P0 mouse retinas with the indicated genotypes were visualized by R26tdTom Cre reporter. Distribution of mTORC1-active cells in the boxed areas was also examined by immunostaining of pS6 and shown in the second row. Proliferation and cell cycle progression of RPCs in the mouse retinas were also examined by 5-ethynyl-2′-deoxyuridine (EdU)/5-chloro-2'-deoxyuridine (CldU) labeling and chasing experiments as described in Materials and methods. (B) R26tdTom-positive Cre-affected cell population in the mouse retinas were quantified by FACS and shown in the graph. Error bars, SD. Numbers of samples analyzed are shown in the graph (four independent litters). (C) RPCs that had progressed from S-phase to G2/M-phase for 3 hr (EdU;pH3-positive); PRs that had been born for 12 hr (EdU;Otx2-positive); and RPCs that had reentered cell cycle for 15 hr (CldU;IdU-positive) in the mouse retinas were counted and shown in the graph. Error bars denote SD (n = 5, five independent litters). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (D) Relative mRNA levels of Hif1a, hexokinase 2 (Hk2), and pyruvate kinase M2 (Pkm2) in P0 mouse retinas with the indicated genotypes were examined by real-time quantitative polymerase chain reaction (RT-qPCR). The y-axis values are relative 2-ΔCt values against those of Tsc1fl/+;Tyrp1- Cre mouse retinas. Error bars, SD (n = 4, three independent litters). (E) Expressions of the indicated proteins in the mouse retinas were examined by Western blot (WB). Relative amounts of the proteins used in each sample were determined by WB detection of β-actin. (F) Relative WB band intensities of the proteins are determined by the ImageJ software and shown in the graph. Error bars, SD (n = 3, three independent litters). (G) Distributions of Pkm2, Tom20, and hypoxyprobe-labeled proteins in the mouse retinas were examined by immunostaining. (H) P0 Tsc1fl/fl;Tyrp1-Cre littermate mice were injected with a chemical inhibitor of glycolysis (2DG) or mitochondrial oxidative phosphorylation (Metformin), and the eye sections of the mice were

Article Snippet: 70079 18 of 27 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Anti- BrdU(CldU) (Rat monoclonal) Novus Biologicals NB500- 169 IHC (1:200) Antibody Anti- BrdU(IdU) (Mouse monoclonal) EXBIO 11–286 C100 IHC (1:200) Antibody Anti- Brn3b (Rabbit polyclonal) Santa Cruz Biotechnology sc- 31989 IHC (1:200) Antibody Anti- Calbindin (Rabbit polyclonal) Swant Inc. CB- 38 IHC (1:200) Antibody Anti- Cdo (Goat polyclonal) R&D Systems AF2429 IHC (1:200) Antibody Anti- c- myc (Mouse monoclonal) Santa Cruz Biotechnology sc40 WB (1:1000) Antibody Anti- Cleaved caspase- 3 (Rabbit polyclonal) Cell Signaling Technology 9661 IHC (1:200) Antibody Anti- Ezrin (Mouse monoclonal) Invitrogen Biotechnology 35–7300 IHC (1:200) Antibody Anti- GFAP (Rabbit polyclonal) Abcam ab48050 IHC (1:200) Antibody Anti- GFP (Chicken polyclonal) Abcam ab13970 IHC (1:2000) Antibody Anti- Glutamine synthase (Rabbit polyclonal) Sigma- Aldrich G- 2781 IHC (1:200) Antibody Anti- Hif1a (Mouse monoclonal) R&D Systems MAB1536 WB (1:1000) Antibody Anti- Hif2α/Epas1 (Rabbit polyclonal) Novus Biologicals NB100- 122 WB (1:1000) Antibody Anti- Hk2 (Rabbit polyclonal) Cell Signaling Technology 2867 WB 1:1,000 IHC (1:200) Antibody Anti- Hydroxy- Hif1alpha (Rabbit polyclonal) Cell Signaling Technology 3434 WB (1:1000) Antibody Anti- M- opsin Merck Millipore AB5405 IHC (1:200) Antibody Anti- Otx2 (Rabbit polyclonal) Abcam ab25985 IHC (1:200) Antibody Anti- Otx2 (Rabbit polyclonal) Abcam ab183951 IHC (1:200) Antibody Anti- Otx2 (Goat polyclonal) R&D Systems AF1979- SP IHC (1:200) Antibody Anti- p21/Cip1 (Mouse monoclonal) Santa Cruz Biotechnology SC817 WB (1:1000) IHC (1:200) Antibody Anti- p53 (Mouse monoclonal) Merck Millipore OP03- 100 WB (1:1000) IHC (1:200) Antibody Anti- Pax6 (Rabbit polyclonal) Covance PRB- 278P IHC (1:200) Antibody Anti- Phospho Smad1/5(S463/465) (Rabbit polyclonal) Invitrogen Biotechnology 700047 IHC (1:200) Antibody Anti- phospho Smad2(ser465/467) (Rabbit polyclonal) - Cell Signaling Technology 18338 IHC (1:200) Antibody Anti- phospho- Histone H3(S10) (pH3; Rabbit polyclonal) Merck Millipore 04–1093 IHC (1:200) Antibody Anti- phospho- S6(S235/236) (pS6; Rabbit polyclonal) Cell Signaling Technology 2211 WB (1:1000) IHC (1:200) Continued Continued on next page Lim et al. eLife 2021;10:e70079.

Techniques: Expressing, Immunostaining, Labeling, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Software, Injection, Phospho-proteomics

Journal: PLoS ONE

Article Title: Adiponectin-Mediated Analgesia and Anti-Inflammatory Effects in Rat

doi: 10.1371/journal.pone.0136819

Figure Lengend Snippet: Real-Time PCR probe, forward and reverse primer sequences for detection of rat cyclophilin, adiponectin, adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2).

Article Snippet: Following blocking, membranes were incubated overnight at 4°C with either adiponectin (rabbit polyclonal; 1:1000; Abcam, UK), AdipoR1 (rabbit polyclonal; 1:1000; Santa Cruz Biotechnology, Inc. USA) or AdipoR2 (goat polyclonal; 1:1000; Santa Cruz Biotechnology, Inc. USA) primary antisera diluted in blocking buffer.

Techniques: Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Adiponectin-Mediated Analgesia and Anti-Inflammatory Effects in Rat

doi: 10.1371/journal.pone.0136819

Figure Lengend Snippet: Photomicrograph shows expression of AdipoR1 and AdipoR2 at 45 kDa, as expected, in a representative control animal. Protein molecular weights are indicated in the size marker (M) lanes.

Article Snippet: Following blocking, membranes were incubated overnight at 4°C with either adiponectin (rabbit polyclonal; 1:1000; Abcam, UK), AdipoR1 (rabbit polyclonal; 1:1000; Santa Cruz Biotechnology, Inc. USA) or AdipoR2 (goat polyclonal; 1:1000; Santa Cruz Biotechnology, Inc. USA) primary antisera diluted in blocking buffer.

Techniques: Expressing, Control, Marker

Journal: EBioMedicine

Article Title: PPARγ maintains the metabolic heterogeneity and homeostasis of renal tubules

doi: 10.1016/j.ebiom.2018.10.072

Figure Lengend Snippet: PPARγ suppresses glycolysis via inhibition of EGFR in PT cells. (A) NRK-52E cells with or without PPARγ depletion were transfected with a control vector or a vector expressing PPARγ. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, HK2, PFKL, GLUT, LDHA. All the experiments data were performed in triplicate at least and the representative imagines were shown. (B, C) The media of serum-starved NRK-52E cells with or without PPARγ depletion and with or without reconstituted expression of PPARγ were collected for analysis of glucose consumption (B) and lactate production(C). The data represent the mean ± SD from n = 3 independent experiments. *** P ≤ .001. (D) NRK-52E cells with or without PPARγ depletion were lysed. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, P-EGFR, Total-EGFR, P-ERK1/2, total-ERK1, P-AKT, Total-AKT. All the experiments data were performed in triplicate at least and the representative imagines were shown. (E) NRK-52E cells with or without PPARγ depletion were treated with or without EGFR inhibitor AG1478 (2 μM) for 48 h. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, P-EGFR, HK2, PFKL, GLUT1, LDHA. All the experiments data were performed in triplicate at least and the representative imagines were shown. (F) NRK-52E cells with or without PPARγ overexpression were treated with or without EGF (100 ng/ml) for 24 h. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, HK2, PFKL, GLUT1, GAPDH, LDHA. All the experiments data were performed in triplicate at least and the representative imagines were shown. (G, H) The media of serum-starved NRK-52E cells with or without PPARγ depletion in the presence or absence of AG1478 (2 μM) for 48 h were collected for analysis of glucose consumption (G) and lactate production (H). The data represent the mean ± SD from n = 3 independent experiments. * P ≤ .05;** P ≤ .01; *** P ≤ .001.

Article Snippet: Subsequently, the membrane was washed with 1 × PBS to remove Ponceau S and blocked with 5% ( w / v ) fat-free milk in TBST at room temperature for 1 h. Then the membrane was probed with primary antibodies (anti-PPARα (1:1000 dilution, Abcam),anti-Glut1 (1:1000 dilution, Abcam), anti-NCC (1:1000 dilution, Abcam), anti-iRhom2(1:500 dilution, Abcam), anti-c-Myc(1:1000 dilution, Abcam) anti-CD28K(1:1000 dilution, Cell Signaling Technology) anti-PFKL(1:1000 dilution, Cell Signaling Technology), anti-β-tubulin(1:2000 dilution, Cell Signaling Technology), anti-EGFR(1:2000 dilution, Cell Signaling Technology), anti-phospho-EGFR(1:2000 dilution, Cell Signaling Technology), anti-phospho-ERK(1:2000 dilution, Cell Signaling Technology), anti-phospho-AKT(1:2000 dilution, Cell Signaling Technology), anti-AKT(1:2000 dilution, Cell Signaling Technology), anti-PPARγ(1:1000 dilution, Proteintech), anti-ACOX1(1:1000 dilution, Proteintech), anti-ACSL1(1:1000 dilution, Proteintech), anti-ACAA1(1:1000 dilution, Proteintech), anti-ACAA2(1:1000 dilution, Proteintech), anti-HADH(1:1000 dilution, Proteintech), anti-HK2(1:1000 dilution, Proteintech), anti-LDHA(1:5000 dilution, Proteintech) anti-EGF(1:1000 dilution, Bioworld), anti-CD36(1:1000 dilution, Bioworld), anti-Podocin(1:1000 dilution, Santa Cruz Biotechnology) and anti-ERK1(1:1000 dilution, Santa Cruz Biotechnology)) at 4 °C overnight, The membrane was then washed with 1 × TBST at room temperature 3 times for 5 min and probed with corresponding second antibody at room temperature for 1 h. The final signals of WB fragments were developed by Chemiluminescent Horseradish Peroxidase (HRP) Substrate Reagent (Millipore, Billerica, MA, USA) and then were detected with ChemiDoc™ XRS+ (Bio-Rad, Hercules, CA, USA).

Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, Western Blot, Over Expression

Journal: EBioMedicine

Article Title: PPARγ maintains the metabolic heterogeneity and homeostasis of renal tubules

doi: 10.1016/j.ebiom.2018.10.072

Figure Lengend Snippet: PPARγ suppresses the glycolysis in PT through iRhom2-mediated downregulation of EGF expression and secretion. (A) Real time PCR analyses of mRNA levels of the indicated gene in NRK-52E cells with or without PPARγ depletion were performed. The data represent the mean ± SD from n = 3 independent experiments. *** P ≤ .0 01. ns represents not significant difference between the indicated sample and the counterpart in the absence of PPARγ depletion. (B) The intracellular and secreted EGF levels of NRK-52E cells with or without PPARγ depletion were analyzed by immunoblotting assay with the indicated antibodies including PPARγ and EGF (left) and ELISA (right), respectively. * P ≤ .05. (C) NRK-52E cells with or without PPARγ depletion were treated with or without EGF neutralizing antibody for 48 h. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, P-EGFR, Total-EGFR. All the experiments data were performed in triplicate at least and the representative imagines were shown. (D) The mRNA levels of PPAR γ and RHBDF2 of NRK-52E cells with or without PPARγ depletion were determined by real time PCR analysis. The data represent the mean ± SD from n = 3 independent experiments. *** P ≤ .001. (E, F) NRK-52E cells with or without PPARγ depletion were transfected with or without a vector expressing iRhom2. Immunoblotting analyses with the indicated antibodies including PPARγ, iRhom2, EGF, P-EGFR, Total-EGFR, P-ERK1/2, Total-ERK1, P-AKT, Total-AKT, HK2, PFKL, GLUT1, LDHA (E) and ELISA analyses of secreted EGF levels (F) were performed. The data represent the mean ± SD from n = 3 independent experiments. ** P ≤ .01. (G) Sequence alignment of the putative PPRE within the human, rat, and mouse RHBDF2 promoters. The varied nucleotides in WT RHBDF2 promoter of different species and mutated nucleotides in the RHBDF2 promoter used for luciferase assay were labeled in red. (H) ChIP analyses of NRK-52E cells were performed with an anti-PPARγ antibody and the primers for the putative PPRE region of RHBDF2 promoter. (I) Luciferase reporter vectors with species-specific or mutated RHBDF2 promoter were co-transfected with the vector expressing PPARγ into HEK293FT cells treated with or without PPARγ ligand Rosiglitazone (TZD) (50 μM). Luciferase reporter analyses were performed. The data represent the mean ± SD from n = 3 independent experiments. ** P ≤ .01; *** P ≤ .001. (J, K) Serum-starved NRK52-E cells with or without PPARγ depletion or iRhom2 overexpression were treated with or without EGF (100 ng/ml) for 24 h. The media were collected for analysis of glucose consumption (j) and lactate production (k). The data represent the mean ± SD from n = 3 independent experiments. * P ≤ .05; ** P ≤ .01.

Article Snippet: Subsequently, the membrane was washed with 1 × PBS to remove Ponceau S and blocked with 5% ( w / v ) fat-free milk in TBST at room temperature for 1 h. Then the membrane was probed with primary antibodies (anti-PPARα (1:1000 dilution, Abcam),anti-Glut1 (1:1000 dilution, Abcam), anti-NCC (1:1000 dilution, Abcam), anti-iRhom2(1:500 dilution, Abcam), anti-c-Myc(1:1000 dilution, Abcam) anti-CD28K(1:1000 dilution, Cell Signaling Technology) anti-PFKL(1:1000 dilution, Cell Signaling Technology), anti-β-tubulin(1:2000 dilution, Cell Signaling Technology), anti-EGFR(1:2000 dilution, Cell Signaling Technology), anti-phospho-EGFR(1:2000 dilution, Cell Signaling Technology), anti-phospho-ERK(1:2000 dilution, Cell Signaling Technology), anti-phospho-AKT(1:2000 dilution, Cell Signaling Technology), anti-AKT(1:2000 dilution, Cell Signaling Technology), anti-PPARγ(1:1000 dilution, Proteintech), anti-ACOX1(1:1000 dilution, Proteintech), anti-ACSL1(1:1000 dilution, Proteintech), anti-ACAA1(1:1000 dilution, Proteintech), anti-ACAA2(1:1000 dilution, Proteintech), anti-HADH(1:1000 dilution, Proteintech), anti-HK2(1:1000 dilution, Proteintech), anti-LDHA(1:5000 dilution, Proteintech) anti-EGF(1:1000 dilution, Bioworld), anti-CD36(1:1000 dilution, Bioworld), anti-Podocin(1:1000 dilution, Santa Cruz Biotechnology) and anti-ERK1(1:1000 dilution, Santa Cruz Biotechnology)) at 4 °C overnight, The membrane was then washed with 1 × TBST at room temperature 3 times for 5 min and probed with corresponding second antibody at room temperature for 1 h. The final signals of WB fragments were developed by Chemiluminescent Horseradish Peroxidase (HRP) Substrate Reagent (Millipore, Billerica, MA, USA) and then were detected with ChemiDoc™ XRS+ (Bio-Rad, Hercules, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Sequencing, Luciferase, Labeling, Over Expression

Journal: PLoS ONE

Article Title: Gli2 Acetylation at Lysine 757 Regulates Hedgehog-Dependent Transcriptional Output by Preventing Its Promoter Occupancy

doi: 10.1371/journal.pone.0065718

Figure Lengend Snippet: ( A ) In vivo acetylation assay in HEK293T cells transfected with plasmids coding for Myc tagged Gli2 and the indicated HATs. Cell extracts were immunoprecipitated with anti-Myc antibody and acetylation was revealed with anti-acetyl lysine antisera. Western blot analysis with anti Myc antibody showed equivalent amounts of Myc-Gli2. Empty, pcDNA3. ( B ) Luciferase assay in HEK293T. Cells were transfected with 12× Gli-Luc and TK renilla reporter vectors, Gli2, p300, TIP60, pCAF where indicated. *p<0.01 vs Empty (pcDNA3). Results are shown as the average ± SD of triplicate experiments (n = 4). ( C ) (Top) ClustalW alignment of the region surrounding lysine 757 of Gli2 in different species. Identical, strongly conservative and weakly conservative aminoacids are indicated by asterisk, colon and dots respectively, according to ClustalW convention. (Bottom) In vivo acetylation assay in HEK293T cells transfected with Myc-Gli2 WT or K757R and p300 where indicated. Acetylation was detected as previously described. ( D ) (Top) Luciferase assay performed in HEK293T cells transfected with 12× Gli-Luc, TK renilla, Myc-Gli2 WT or the acetylation mimetic mutant Myc-Gli2 K757Q. *p<0.01 vs Empty; **p<0.05 vs WT. Results are shown as the average ± SD of triplicate experiments (n = 3). (Bottom) Western blot analysis of Myc-Gli2 WT and K757Q expression levels.

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against acetylated lysine (1∶1000, Upstate, 06933), goat anti Gli2 (1∶1000, R&D systems), anti-Gli2 (C-10) (Santa Cruz, sc-271786), mouse anti Myc-HRP (1∶ 2000, Sigma), mouse anti-Myc (1∶1000, Sigma, M4439), anti Acetyl-Gli2(Lys757) (1∶1000) anti-Tubulin (1∶1000, SantaCruz sc-8035), anti-CREB (1∶2000, gift from M. Montminy, Salk Institute La Jolla, CA).

Techniques: In Vivo, Acetylation Assay, Transfection, Immunoprecipitation, Western Blot, Luciferase, Mutagenesis, Expressing

Journal: PLoS ONE

Article Title: Gli2 Acetylation at Lysine 757 Regulates Hedgehog-Dependent Transcriptional Output by Preventing Its Promoter Occupancy

doi: 10.1371/journal.pone.0065718

Figure Lengend Snippet: ( A ) K757 acetylation of wild type Myc tagged Gli2 and K757R mutant in HEK293T cells. Cells extract were immunoprecipitated with anti-Myc antibody and acetylation was detected by anti acetyl-Gli2(Lys757) specific antisera. Western blot analysis with anti Myc antibody showed equivalent amounts of Myc-Gli2. ( B ) Acetylation of endogenous Gli2 in NIH3T3 cells, treated with SAG or DMSO for 24 hours. Acetylation was detected with anti acetyl-Gli2(Lys757) specific antisera. ( C ) (Top) Luciferase assay in NIH3T3 cells transfected with 8× Gli-Luc reporter, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty. Results are shown as the average ± SD of triplicate experiments (n = 3). (Bottom) Gli1 mRNA levels normalized with the housekeeping HPRT mRNA in NIH3T3 cells transfected with Myc-Gli2 WT and K757R mutant. ( D ) Transcriptional activity of Myc-Gli2 WT and K757R mutant in response to SAG treatment: Ptch1 mRNA levels (QPCR), normalized with the housekeeping GAPDH mRNA in 24 hours SAG-treated NIH3T3 cells, transfected with Empty vector, Myc tagged Gli2 wild type and K757R mutant. *p<0.01 vs Empty, **p<0.05 vs DMSO; Results are shown as the average ± SD of triplicate experiments (n = 3).

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against acetylated lysine (1∶1000, Upstate, 06933), goat anti Gli2 (1∶1000, R&D systems), anti-Gli2 (C-10) (Santa Cruz, sc-271786), mouse anti Myc-HRP (1∶ 2000, Sigma), mouse anti-Myc (1∶1000, Sigma, M4439), anti Acetyl-Gli2(Lys757) (1∶1000) anti-Tubulin (1∶1000, SantaCruz sc-8035), anti-CREB (1∶2000, gift from M. Montminy, Salk Institute La Jolla, CA).

Techniques: Mutagenesis, Immunoprecipitation, Western Blot, Luciferase, Transfection, Activity Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: Gli2 Acetylation at Lysine 757 Regulates Hedgehog-Dependent Transcriptional Output by Preventing Its Promoter Occupancy

doi: 10.1371/journal.pone.0065718

Figure Lengend Snippet: Immunohistochemical staining of Gli1(upper-right), acetyl-Gli1(Lys518) (bottom-left), acetyl-GLi2(Lys757) (bottom-right), or Hematoxylin (upper-left) in a 6-day-old mouse cerebellum.

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against acetylated lysine (1∶1000, Upstate, 06933), goat anti Gli2 (1∶1000, R&D systems), anti-Gli2 (C-10) (Santa Cruz, sc-271786), mouse anti Myc-HRP (1∶ 2000, Sigma), mouse anti-Myc (1∶1000, Sigma, M4439), anti Acetyl-Gli2(Lys757) (1∶1000) anti-Tubulin (1∶1000, SantaCruz sc-8035), anti-CREB (1∶2000, gift from M. Montminy, Salk Institute La Jolla, CA).

Techniques: Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Gli2 Acetylation at Lysine 757 Regulates Hedgehog-Dependent Transcriptional Output by Preventing Its Promoter Occupancy

doi: 10.1371/journal.pone.0065718

Figure Lengend Snippet: ( A ) Cellular fractionation of NIH3T3 cells shows no difference in the localization of Myc tagged Gli2 wild type (WT), K575R and K575Q mutants. Purity of fractionation documented by tubulin (cytoplasmic, Cyto) and CREB (nuclear, Nu) staining. ( B ) Promoter occupancy of Gli2 is prevented by K757 acetylation. HEK293T cells were transfected with Myc tagged Gli2 wild type (WT), K757R, K757Q and Empty vectors and Chromatin Immunoprecipitation (ChIP) was carried out. Quantitative real-time PCR was performed using primers encompassing the Gli-BS of human Ptch1 promoter (bottom, schematic representation). Results are indicated as fold difference, relative to Empty (pcDNA3) control. *p<0.05 WT vs Empty, **p<0.05 K757Q vs WT. Results are shown as the average ± SD of triplicate experiments (n = 4). Western blot analysis with anti Myc antibody showing equivalent amounts of Myc-Gli2 WT and mutants. Empty, pcDNA3.

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against acetylated lysine (1∶1000, Upstate, 06933), goat anti Gli2 (1∶1000, R&D systems), anti-Gli2 (C-10) (Santa Cruz, sc-271786), mouse anti Myc-HRP (1∶ 2000, Sigma), mouse anti-Myc (1∶1000, Sigma, M4439), anti Acetyl-Gli2(Lys757) (1∶1000) anti-Tubulin (1∶1000, SantaCruz sc-8035), anti-CREB (1∶2000, gift from M. Montminy, Salk Institute La Jolla, CA).

Techniques: Cell Fractionation, Fractionation, Staining, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Gli2 Acetylation at Lysine 757 Regulates Hedgehog-Dependent Transcriptional Output by Preventing Its Promoter Occupancy

doi: 10.1371/journal.pone.0065718

Figure Lengend Snippet: Following Hh/Ptch interaction, Smo triggers a signaling cascade leading to Gli2 deacetylation and to the inhibition of the βTrCP-regulated balance between Gli2R and full length Gli2 (Gli2A). Both events contributes to the early signal-dependent activation of the Hh pathway. Once activated, Gli2 promotes transcription of Gli1, whose activity is also regulated by Hh-induced HDAC1-mediated deacetylation, thus generating a positive feedback loop (late activation).

Article Snippet: The following antibodies were used: rabbit polyclonal antibody against acetylated lysine (1∶1000, Upstate, 06933), goat anti Gli2 (1∶1000, R&D systems), anti-Gli2 (C-10) (Santa Cruz, sc-271786), mouse anti Myc-HRP (1∶ 2000, Sigma), mouse anti-Myc (1∶1000, Sigma, M4439), anti Acetyl-Gli2(Lys757) (1∶1000) anti-Tubulin (1∶1000, SantaCruz sc-8035), anti-CREB (1∶2000, gift from M. Montminy, Salk Institute La Jolla, CA).

Techniques: Inhibition, Activation Assay, Activity Assay

Journal: Cancers

Article Title: KIF2C Facilitates Tumor Growth and Metastasis in Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers15051502

Figure Lengend Snippet: Analysis after sequencing and the results of cell cycle detection, real-time PCR, and Western blot validation. ( A ) The results of the GO analysis, in which the functions related to the cell cycle and mitosis are the most noteworthy. ( B ) Cell cycle detection of the transfected cell lines in each group, during which the S phase of the sh-KIF2C and KIF2C groups increased (*** p < 0.001, **** p < 0.0001). ( C ) Heat map of the differential genes in the overexpression and NC groups. ( D ) The results of the KEGG analysis, where the Nod-like receptor and cell cycle signaling pathways are perhaps the main pathways in which KIF2C plays a role. ( E ) The results of the real-time PCR detection of some differential genes, showing that changes in KIF2C do affect the expression of other genes. ( F ) Correlation analysis between KIF2C and CDC20 in the TCGA database (Red box and gray box represent cancer tissue and paracancerous tissue respectively). ( G ) Western blot analysis of the IL-1β, IL-18, and CDC20 levels (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The antibodies used in this study were as follows: KIF2C (1:1000, Sigma, Darmstadt, Germany), IL-1β (1:1000, Elabscience, Wuhan, China), IL-18 (1:1000, Elabscience, Wuhan, China), CDC20 (1:2000, Proteintech, Chicago, USA), and GAPDH: (1:5000, Proteintech, Chicago, USA).

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Over Expression, Expressing

Journal: Journal of Pediatric Hematology/Oncology

Article Title: Chemotherapy-induced Alteration of SDF-1/CXCR4 Expression in Bone Marrow–derived Mesenchymal Stem Cells From Adolescents and Young Adults With Acute Lymphoblastic Leukemia

doi: 10.1097/mph.0000000000000220

Figure Lengend Snippet: FIGURE 3. Secretion and expression of SDF-1 in BM-MSCs. A, SDF-1 level was measured by enzyme-linked immunosorbent assay. (a) The bone marrow plasma level of SDF-1 in patients with ALL was significantly increased compared with that in healthy donors and ALL-c (*P < 0.05; n = 10). (b), The same difference was found when SDF-1 levels were measured in the supernatants of MSC cultures after incubation for 72 hours. B, Western blotting was performed to quantify total SDF-1 in the lysates of MSCs. SDF-1 expression in the MSCs of ALL was significantly higher than that in the MSCs of healthy donors and ALL-c. C, The difference was also found at the transcriptional level by real-time PCR (*P < 0.05, n = 10). D, Surface-CD26 expression on the MSCs from ALL patients measured by flow cytometry was significantly lower than that in healthy donors and ALL-c (*P < 0.05; n = 10). E, Total CD26 was measured by Western blot in the MSCs. The fold difference was shown among the 3 types of MSCs. F, The same difference was also found at the transcriptional level (*P < 0.05; n = 10). ALL indicates acute lymphoblastic leukemia; BM-MSC, bone marrow–derived mesenchymal stem cell; GADPH, glyceraldehyde- 3-phosphate dehydrogenase; PCR, polymerase chain reaction.

Article Snippet: Membranes were hybridized with the following primary antibodies: rabbit anti-human CXCR4 (1:1000; Abcam), goat antihuman SDF-1(1:500; R&D Systems), goat anti-human CD26 (1:1000; R&D Systems), and mouse antibody to glyceraldehyde-3-phosphate dehydrogenase (1:4000; Santa Cruz).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Flow Cytometry, Derivative Assay, Polymerase Chain Reaction

Journal: Journal of the National Cancer Institute

Article Title: Combined treatment strategies for microtubule stabilizing agent-resistant tumors.

doi: 10.1093/jnci/dju504

Figure Lengend Snippet: Figure 1. Regulation of vascular endothelial growth factor (VEGF)–mRNA-expression (A) and VEGF secretion (B) by patupilone and everolimus. A549EpoB40 cells were treated with patupilone (0.2 nM, 24 hours) and/or everolimus (2.5 nM, one hour) and cultured under normoxic and hypoxic (0.2%) conditions. VEGF mRNA was deter- mined 24 hours after starting hypoxic conditions by real-time polymerase chain reaction. Supernatants were collected and analyzed for VEGF protein levels by enzyme- linked immunosorbent assay. Data are means of at least three independent experiments. Error bars represent standard deviations. C) Patupilone- and everolimus- treated A549EpoB40 cell lysates were tested against the phosphorylated form of p70-S6Kinase and HIF-1α subunit by western blotting. Cells were preincubated with patupilone and everolimus for half an hour followed by additional incubation for four hours under normoxic or hypoxic conditions and cell lysis. Picture shows one representative experiment out of three experiments.

Article Snippet: Antibody detection was achieved by enhanced chemoluminescence (Amersham, Piscataway, NJ) using a polyclonal rabbit anti– HIF-1α subunit antibody (Santa Cruz Biotechnology, #sc-10790, 1:1000), a rabbit polyclonal anti–phospho-P70-S6kinase antibody (Cell Signalling Technology, #9205, 1:1000), a rabbit polyclonal anti–P70-S6kinase antibody (Cell Signalling Technology, #9202, 1:1000) and a mouse monoclonal anti–β-actin antibody (Sigma Aldrich, #A5441, 1:1000).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Lysis

Journal: Journal of the National Cancer Institute

Article Title: Combined treatment strategies for microtubule stabilizing agent-resistant tumors.

doi: 10.1093/jnci/dju504

Figure Lengend Snippet: Figure 4. Response to treatment with patupilone, everolimus, and bevacizumab alone and in combination. A) Microvessel density. B) mTOR activity. C) Tumor cell proliferation. Mice carrying A549EpoB40-derived xenografts (n = 3 per group) were treated as described, and tumors were collected 24 hours after the last treatment with everolimus and bevacizumab alone or in combination with patupilone. Tumors from patupilone alone–treated mice were collected 24 hours after the last dose of the corresponding combined treatment modality. Tumors were harvested, formalin fixed and stained for CD31, phospho-P70-S6Kinase, and MIB1/Ki-67. Error bars represent standard deviations. Data were analyzed by one-way analysis of variance on replicate tumor samples (n = 6–9), and pair-wise analysis was performed using the Tukey Test. Relevant comparisons are given in the text and figures. All statistical tests were two-sided. MVD = microvessel density.

Article Snippet: Antibody detection was achieved by enhanced chemoluminescence (Amersham, Piscataway, NJ) using a polyclonal rabbit anti– HIF-1α subunit antibody (Santa Cruz Biotechnology, #sc-10790, 1:1000), a rabbit polyclonal anti–phospho-P70-S6kinase antibody (Cell Signalling Technology, #9205, 1:1000), a rabbit polyclonal anti–P70-S6kinase antibody (Cell Signalling Technology, #9202, 1:1000) and a mouse monoclonal anti–β-actin antibody (Sigma Aldrich, #A5441, 1:1000).

Techniques: Activity Assay, Derivative Assay, Staining

Journal: Journal of the National Cancer Institute

Article Title: Combined treatment strategies for microtubule stabilizing agent-resistant tumors.

doi: 10.1093/jnci/dju504

Figure Lengend Snippet: Figure 5. Representative tumor sections depicting microvessel density, high magnification images of representative vessels, mTOR activity, and tumor cell proliferation in response to treatment. Mice carrying A549EpoB40-derived xenografts (n = 3 per group) were treated as described, and tumors were collected 24 hours after the last treatment with everolimus and bevacizumab alone or in combination with patupilone. Tumors from patupilone alone–treated mice were collected 24 hours after the last dose of the corresponding combined treatment modality. Tumors were harvested, formalin fixed and stained for CD31, phospho-P70-S6Kinase, and MIB1/Ki-67. Arrows indicate microvessels. Scale bars for all pictures correspond to 100 µm.

Article Snippet: Antibody detection was achieved by enhanced chemoluminescence (Amersham, Piscataway, NJ) using a polyclonal rabbit anti– HIF-1α subunit antibody (Santa Cruz Biotechnology, #sc-10790, 1:1000), a rabbit polyclonal anti–phospho-P70-S6kinase antibody (Cell Signalling Technology, #9205, 1:1000), a rabbit polyclonal anti–P70-S6kinase antibody (Cell Signalling Technology, #9202, 1:1000) and a mouse monoclonal anti–β-actin antibody (Sigma Aldrich, #A5441, 1:1000).

Techniques: Activity Assay, Derivative Assay, Staining

Journal: Journal of the American Heart Association

Article Title: Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex

doi: 10.1161/jaha.123.030460

Figure Lengend Snippet: Figure 1. The epigenetic factors DNMT1 and MeCP2 and the transcriptional factor REST are involved in the negative modulation of Ncx1 mRNA and protein expression. A and B, qRT-PCR for DNMT1 and MECP2 transcripts in SH-SY5Y cells transfected for 48 hours with siDNMT1 or siMeCP2 at a final concentration of 50 nM. An siCTL was used as scrambled. *P≤0.05 vs siCTL by Student’s t test (n=3). C, Ncx1 mRNA and protein expression in SH-SY5Y cells transfected for 48 hours with siCTL, siDNMT1, siMeCP2, and siREST. *P≤0.05 vs siCTL by 1-way ANOVA analysis followed by Tukey’s post hoc test (n=3/4). D and E, qRT-PCR for DNMT1 and MECP2 transcripts in SH-SY5Y transfected for 48 hours with vector overexpressing DNMT1 or MeCP2. pcDNA3.1 vector was used as EV. *P≤0.05 vs EV by Student’s t test (n=3). F, Ncx1 mRNA and protein expression in SH-SY5Y cells transfected for 48 hour with EV, DNMT1, MeCP2, or REST plasmids. *P≤0.05 vs EV by 1-way ANOVA analysis followed by Tukey’s post hoc test (n=3/4). CTL indicates control; DNMT1, DNA-methyltransferase-1; EV, empty vector; MeCP2, methyl-CpG binding protein 2; NCX1, sodium/calcium exchanger 1; qRT-PCR, quantitative real time polymerase chain reaction; REST, repressor element 1-silencing transcription factor; si, small interfering; and siCTL, control siRNA.

Article Snippet: Chromatin lysates were incubated overnight with 3 μg of antibody for DNMT1 (Mouse mAb, cod: sc- 271 729 Santa Cruz Biotechnology, dilution 1:500), MeCP2 (Rabbit mAb, cod: 3456 Cell Signaling, dilution 1:1000), REST, HDAC1, and HDAC2.3 Normal rabbit IgG was used as negative control.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Concentration Assay, Plasmid Preparation, Control, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association

Article Title: Stroke Causes DNA Methylation at Ncx1 Heart Promoter in the Brain Via DNMT1/MeCP2/REST Epigenetic Complex

doi: 10.1161/jaha.123.030460

Figure Lengend Snippet: Figure 4. DNMT1 and MeCP2 bind Ht, but not Br promoter sequence, in the temporoparietal cortex 24 hours after tMCAO. Chromatin immunoprecipitation with anti-DNMT1 (A), anti-MeCP2 (B) anti-REST (C), anti-HDAC1 (D), anti-HDAC2 (E), antibodies followed by qPCR of Ncx1 brain promoter (Br) (white columns) and Ncx1 heart promoter (Ht) (gray columns) in peri-ischemic cortex of mice euthanized 12 or 24 hours after tMCAO. IgG was used as negative control. *P≤0.05 vs Sham immunopreciptated with IgG antibody by 1-way ANOVA analysis followed by Tukey’s post hoc test (n=3). DNMT1 indicates DNA-methyltransferase-1; HDAC, histone deacetylase; IP, immunoprecipitation; MeCP2, methyl-CpG binding protein 2; NCX1, sodium/calcium exchanger 1; qRT-PCR, quantitative real time polymerase chain reaction; REST, repressor element 1-silencing transcription factor; and tMCAO, transient middle cerebral artery occlusion.

Article Snippet: Chromatin lysates were incubated overnight with 3 μg of antibody for DNMT1 (Mouse mAb, cod: sc- 271 729 Santa Cruz Biotechnology, dilution 1:500), MeCP2 (Rabbit mAb, cod: 3456 Cell Signaling, dilution 1:1000), REST, HDAC1, and HDAC2.3 Normal rabbit IgG was used as negative control.

Techniques: Sequencing, Chromatin Immunoprecipitation, Negative Control, Histone Deacetylase Assay, Immunoprecipitation, Binding Assay, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Frontiers in Physiology

Article Title: Investigation of the Lipid-Lowering Effect of Vitamin C Through GSK-3β/β-Catenin Signaling in Zebrafish

doi: 10.3389/fphys.2018.01023

Figure Lengend Snippet: Real-time quantitative PCR primers for the genes of zebrafish.

Article Snippet: Antibodies directed against β-catenin (Cat. No. 8480, 1:1000), GSK-3β (Cat. No. 12456, 1:1000), Phospho-GSK-3β (Ser9) (Cat. No. 9323, 1:1000), and Phospho-β-Catenin (Ser675) (Cat. No. 4176, 1:1000) were purchased from Cell Signaling Technology Inc. β-actin (sc-1615, 1:10000) was purchased from Santa-Cruz Inc. Densitometry analyses were performed with the Image J software (National Institutes of Health).

Techniques: Real-time Polymerase Chain Reaction

Journal: Frontiers in Physiology

Article Title: Investigation of the Lipid-Lowering Effect of Vitamin C Through GSK-3β/β-Catenin Signaling in Zebrafish

doi: 10.3389/fphys.2018.01023

Figure Lengend Snippet: Effect of VC on the mRNA expression in the muscle of zebrafish. (A) : GSK-3β; (B) : β-catenin; (C) : C/EBPα; (D) : FAS. Values are expressed as means ± s.e.m. ( n = 4). Statistically significant differences are denoted by different letters ( P < 0.05).

Article Snippet: Antibodies directed against β-catenin (Cat. No. 8480, 1:1000), GSK-3β (Cat. No. 12456, 1:1000), Phospho-GSK-3β (Ser9) (Cat. No. 9323, 1:1000), and Phospho-β-Catenin (Ser675) (Cat. No. 4176, 1:1000) were purchased from Cell Signaling Technology Inc. β-actin (sc-1615, 1:10000) was purchased from Santa-Cruz Inc. Densitometry analyses were performed with the Image J software (National Institutes of Health).

Techniques: Expressing

Journal: Frontiers in Physiology

Article Title: Investigation of the Lipid-Lowering Effect of Vitamin C Through GSK-3β/β-Catenin Signaling in Zebrafish

doi: 10.3389/fphys.2018.01023

Figure Lengend Snippet: Effect of GSK-3β RNA interference on the mRNA expression in the muscle of zebrafish. (A) : GSK-3β; (B) : β-catenin; (C) : C/EBPα; (D) : FAS. Values are expressed as means ± s.e.m. ( n = 3). Statistically significant differences are denoted by different letters ( P < 0.05).

Article Snippet: Antibodies directed against β-catenin (Cat. No. 8480, 1:1000), GSK-3β (Cat. No. 12456, 1:1000), Phospho-GSK-3β (Ser9) (Cat. No. 9323, 1:1000), and Phospho-β-Catenin (Ser675) (Cat. No. 4176, 1:1000) were purchased from Cell Signaling Technology Inc. β-actin (sc-1615, 1:10000) was purchased from Santa-Cruz Inc. Densitometry analyses were performed with the Image J software (National Institutes of Health).

Techniques: Expressing

Journal: Frontiers in Physiology

Article Title: Investigation of the Lipid-Lowering Effect of Vitamin C Through GSK-3β/β-Catenin Signaling in Zebrafish

doi: 10.3389/fphys.2018.01023

Figure Lengend Snippet: Effect of GSK-3β RNA interference on glycerol, TG, and FAS activity in the muscle of zebrafish. (A) : glycerol content; (B) : TG content; (C) : FAS activity. Values are expressed as means ± s.e.m. ( n = 3). Statistically significant differences are denoted by different letters ( P < 0.05).

Article Snippet: Antibodies directed against β-catenin (Cat. No. 8480, 1:1000), GSK-3β (Cat. No. 12456, 1:1000), Phospho-GSK-3β (Ser9) (Cat. No. 9323, 1:1000), and Phospho-β-Catenin (Ser675) (Cat. No. 4176, 1:1000) were purchased from Cell Signaling Technology Inc. β-actin (sc-1615, 1:10000) was purchased from Santa-Cruz Inc. Densitometry analyses were performed with the Image J software (National Institutes of Health).

Techniques: Activity Assay

Journal: Frontiers in Physiology

Article Title: Investigation of the Lipid-Lowering Effect of Vitamin C Through GSK-3β/β-Catenin Signaling in Zebrafish

doi: 10.3389/fphys.2018.01023

Figure Lengend Snippet: Effect of VC on the mRNA expression in ZF4 cells. (A) : GSK-3β; (B) : β-catenin; (C) : C/EBPα; (D) : FAS. Values are expressed as means ± s.e.m. ( n = 3). Statistically significant differences are denoted by asterisk ( P < 0.05).

Article Snippet: Antibodies directed against β-catenin (Cat. No. 8480, 1:1000), GSK-3β (Cat. No. 12456, 1:1000), Phospho-GSK-3β (Ser9) (Cat. No. 9323, 1:1000), and Phospho-β-Catenin (Ser675) (Cat. No. 4176, 1:1000) were purchased from Cell Signaling Technology Inc. β-actin (sc-1615, 1:10000) was purchased from Santa-Cruz Inc. Densitometry analyses were performed with the Image J software (National Institutes of Health).

Techniques: Expressing

Journal: Frontiers in Physiology

Article Title: Investigation of the Lipid-Lowering Effect of Vitamin C Through GSK-3β/β-Catenin Signaling in Zebrafish

doi: 10.3389/fphys.2018.01023

Figure Lengend Snippet: Effect of VC on the immunofluorescence of GSK-3β in the ZF4 cells of zebrafish. (A,D,G) : GSK-3β, green; (B,E,H) : nucleus, blue; (C,F,I) : green and blue merger. Bar: 37.5 μm.

Article Snippet: Antibodies directed against β-catenin (Cat. No. 8480, 1:1000), GSK-3β (Cat. No. 12456, 1:1000), Phospho-GSK-3β (Ser9) (Cat. No. 9323, 1:1000), and Phospho-β-Catenin (Ser675) (Cat. No. 4176, 1:1000) were purchased from Cell Signaling Technology Inc. β-actin (sc-1615, 1:10000) was purchased from Santa-Cruz Inc. Densitometry analyses were performed with the Image J software (National Institutes of Health).

Techniques: Immunofluorescence

Journal: Frontiers in Physiology

Article Title: Investigation of the Lipid-Lowering Effect of Vitamin C Through GSK-3β/β-Catenin Signaling in Zebrafish

doi: 10.3389/fphys.2018.01023

Figure Lengend Snippet: Effect of VC on the protein expression in the ZF4 cells of zebrafish. (A) Protein bands of western blot; (B) Effect of VC on GSK-3β; (C) Effect of VC on β-catenin; (D) Effect of VC on the phosphorylation level of GSK-3β; (E) Effect of VC on the phosphorylation level of β-catenin. Values are expressed as means ± s.e.m. ( n = 3). Statistically significant differences are denoted by different letters ( P < 0.05).

Article Snippet: Antibodies directed against β-catenin (Cat. No. 8480, 1:1000), GSK-3β (Cat. No. 12456, 1:1000), Phospho-GSK-3β (Ser9) (Cat. No. 9323, 1:1000), and Phospho-β-Catenin (Ser675) (Cat. No. 4176, 1:1000) were purchased from Cell Signaling Technology Inc. β-actin (sc-1615, 1:10000) was purchased from Santa-Cruz Inc. Densitometry analyses were performed with the Image J software (National Institutes of Health).

Techniques: Expressing, Western Blot, Phospho-proteomics

Journal: Pharmacological research

Article Title: Ablation of endothelial Pfkfb3 protects mice from acute lung injury in LPS-induced endotoxemia

doi: 10.1016/j.phrs.2019.104292

Figure Lengend Snippet: (A-C) Real time-PCR analysis of ICAM-1 (A), VCAM-1 (B), and E-selectin (C) mRNA levels in HPAECs treated with 1 μg/mL LPS for 4 h following transfection with siCTRL or siPFKFB3 for 48 hours. n = 6. (D) Western blot analysis of PFKFB3 and ICAM-1 protein expression and densitometric quantification of ICAM-1 in HPAECs. HPAECs were transfected with siCTRL or siPFKFB3 for 48 hours followed by 1 μg/ml LPS treatment for 4 hours. n = 5. (E) Representative images and quantification of monocyte adhesion to HPAECs transfected with siCTRL or siPFKFB3 and treated with 1 μg/mL LPS for 4 h. n = 3. (F) Western blot analysis and densitometric quantification of ICAM-1, VCAM-1, p65, and p-p65 protein expression in HPAECs treated with LPS. HPAECs were pretreated with 20 μM 3PO for 30 min, and then treated with 1 μg/mL LPS for 4 h. n = 5. (G) Representative images of p65 immunostaining in HPAECs transfected with siCTRL or siPFKFB3 followed by 1 μg/mL LPS treatment for 4 hours. (H) Western blot analysis and densitometric quantification of PFKFB3, ICAM-1, VCAM-1, p65, and p-p65 protein expression in HPAECs after PFKFB3 overexpression with or without p65 inhibitor, Bay or PDTC, treatment. n = 5. Data are represented as means ± SEM. Statistical significance was determined by one-way ANOVA followed by Bonferroni test. *p < 0.05 was considered significant, ** p < 0.01, *** p < 0.001.

Article Snippet: Following electrophoresis, blotting, and blocking in 5% skim milk, blots were probed with the following primary antibodies: PFKFB3 (1:1000, Cat. No. ab181861, abcam), ICAM-1 (1:1000, Cat. No. sc- 8314, Santa Cruz Biotechnology for human; 1:1000, Cat. No. ab25375, Abcam for mice), VCAM-1 (1:1000, Cat. No. 13662, CST, Danvers, MA, USA), p65 (1:1000, Cat. No. 8242, CST), and p-p65 (1:1000, Cat. No. 3033, CST).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Western Blot, Expressing, Immunostaining, Over Expression

Journal: Nature Communications

Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

doi: 10.1038/s41467-024-45961-1

Figure Lengend Snippet: a Western blots of OMV-stimulated HeLa cells show specific autophagosome/lysosome-dependent degradation of mitochondrial marker proteins (TOM20 and TIM23), but not of Golgi (GM130) or endoplasmic reticulum (PDI). Western blots are representative of 3 independent experiments. b DiO-labeled gonococcal OMVs from strain ATCC 49226 colocalize with MitoBright-labeled mitochondria in HeLa cells. Scale bar, 5 μm. c Live HeLa cell microscopy and 3D image reconstruction shows OMVs remain associated with Cascade Blue-labeled endosomes when delivered to MitoBright-labeled mitochondria. Scale bar, 5 μm. The fluorescence colocalization profile of the line is shown. d Gonococcal OMVs from strain ATCC 49226 dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P < 10 −15 . e TEM of HeLa cells stimulated with gonococcal OMVs from strains ATCC 49226 and ZJXSH86 show mitochondrial disruption and capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, one-way ANOVA with post-hoc Tukey test for mitochondrial disruption (Mock-ATCC 49226 OMVs: P = 2 × 10 −11 ; Mock-ZJXSH86 OMVs: P = 2 × 10 −11 ), Kruskal–Wallis with posthoc Dunn test for mitochondria in mitophagy-like structures (Mock-ATCC 49226 OMVs: P = 1 × 10 −6 ; Mock-ZJXSH86 OMVs: P = 7 × 10 −6 ). f Quantitative real-time PCR showing a reduced mitochondrial to genomic DNA ratio in ATCC 49226 OMV-stimulated HeLa cells. Data are mean ± s.d.; n = 3 independent biological replicates, one-way ANOVA with post-hoc Tukey test. g Increased HSP60 and LAMP1 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, unpaired two-tailed t -test, P = 6 × 10 −9 . h Increased HSP60 and LC3 colocalization in ATCC 49226 OMV-stimulated HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, two-tailed Mann–Whitney test (LC3 puncta Mock-OMVs: P = 7 × 10 −12 ; LC3 colocalized HSP60 Mock-ZJXSH86 OMVs: P = 4 × 10 −15 ). Cells in d , e , g , h are from 3 independent experiments. Images in b , c are representative of 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence staining or Western analysis, the following antibodies were used: Rabbit anti-LC3A/B (D3U4C) monoclonal antibodies (CST, #12741, Western 1:1000, immunofluorescence 1:200); Rabbit anti-LAMP1 (D2D11) monoclonal antibodies (CST, #9091, immunofluorescence 1:200); Rabbit anti-HA (C29F4) monoclonal antibodies (CST, #3724, Western 1:1000); Rabbit anti-His (D3I1O) monoclonal antibodies (CST, #12698, Western 1:1000, immunofluorescence 1:200); Rabbit anti-TOM20 (D8T4N) monoclonal antibodies (CST, #42406, Western 1:1000, immunofluorescence 1:200); Rabbit anti-p62 (D6M5X) monoclonal antibodies (CST, #23214, immunofluorescence 1:200); Mouse anti-FLAG (M2) monoclonal antibodies (Sigma, #F1804, Western 1:1000); Rabbit anti-TIM23 polyclonal antibodies (Proteintech, #11123-1-AP, Western 1:1000); Rabbit anti-p62 polyclonal antibodies (Proteintech, #18420-1-AP, Western 1:1000); Rabbit anti-p62 (PS00-61) monoclonal antibodies (Huabio, #HA721171, Western 1:1000); Rabbit anti-PINK1 polyclonal antibodies (Proteintech, #23274-1-AP, Western 1:500); Mouse anti-β-Tubulin polyclonal antibodies (Proteintech, #10068-1-AP, Western 1:1000); Mouse anti-HSP60 (H-1) monoclonal antibodies (Santa Cruz, #sc-13115, immunofluorescence 1:200); Rabbit anti-RNF213 polyclonal antibodies (Merck, #HPA003347, immunofluorescence 1:100, Western 1:500); Rabbit anti-PDI polyclonal antibodies (Proteintec, #11245-1-AP, Western 1:500); Rabbit anti-GM130 polyclonal antibodies (Beyotime, #AF7005, Western 1:500); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Beyotime, #A0208, Western 1:1000); HRP-conjugated goat anti-mouse IgG (H + L) secondary antibodies (Beyotime, #A0216, Western 1:1000); Goat anti-rabbit IgG (H + L) Alexa Fluor 488 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11008, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 405 cross-adsorbed secondary antibodies (Thermo Fisher, #A-31553, immunofluorescence 1:200); Goat anti-rabbit IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11012, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11032, immunofluorescence 1:200).

Techniques: Western Blot, Marker, Labeling, Microscopy, Fluorescence, Membrane, Two Tailed Test, Disruption, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Nature Communications

Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

doi: 10.1038/s41467-024-45961-1

Figure Lengend Snippet: a TEM of HeLa cells expressing gonococcal PorB from strain ATCC 49226 show mitochondrial capture in mitophagy-like structures. Data are mean ± s.d.; n = 21 cells, two-tailed Mann–Whitney test, P = 2 × 10 −10 . b Western blots showing degradation of mitochondrial proteins TOM20 and TIM23 in HeLa cells expressing gonococcal PorB from strains ATCC 49226 and ZJXSH86, but not for PorB from Neisseria mucosa . c Western blots showing that gonococcal OMVs expressing PorB from N. mucosa lost the ability to induce degradation of TOM20 and TIM23 in HeLa cells. d Gonococcal OMVs expressing PorB from N. mucosa lost the ability to dissipate the mitochondrial membrane potential (MMP) in HeLa cells. Scale bar, 5 μm. Data are mean ± s.d.; n = 55 cells from 4 independent experiments, Kruskal–Wallis with posthoc Dunn test (Mock-N.g. PorB: P = 8 × 10 −13 ; N.g. PorB-N.m. PorB: P < 10 −15 ). e Flow cytom e try analysis of TMRM fluorescence intensity in HeLa cells stimulated with gonococcal OMVs expressing gonococcal PorB or PorB from N. mucosa . f Gonococcal PorB structure (pdb entry 4AUI) and sequence of PorB from strain ATCC 49226. Lysines are indicated in blue, or in magenta when located within the PorB channel and associated with ATP (orange) binding. g Western blots showing reduced degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q. h Reduced HSP60 and LC3 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 2 × 10 −9 ; WT PorB-PorB K117Q: P = 1 × 10 −5 ). i Reduced HSP60 and LAMP1 colocalization in HeLa cells expressing PorB K117Q compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 51 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; Empty-PorB K117Q: P = 9 × 10 −8 ). j Western blots of HeLa 4KO cells showing p62 expression restores PorB K117Q-dependent degradation of TOM20 and TIM23, while expression of OPTN or NDP52 restores PorB K5-dependent degradation of TOM20 and TIM23. k Western blots showing impaired degradation of TOM20 and TIM23 in HeLa cells expressing PorB K117Q, K5 or K null . l Western blots showing that knock-down of PINK1 in HeLa cells inhibits PorB K5-dependent degradation of TOM20 and TIM23, but not for PorB K117Q. Cells in a , h , i are from 3 independent experiments. Western blots in b , c , g , j , k , l are representative of 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence staining or Western analysis, the following antibodies were used: Rabbit anti-LC3A/B (D3U4C) monoclonal antibodies (CST, #12741, Western 1:1000, immunofluorescence 1:200); Rabbit anti-LAMP1 (D2D11) monoclonal antibodies (CST, #9091, immunofluorescence 1:200); Rabbit anti-HA (C29F4) monoclonal antibodies (CST, #3724, Western 1:1000); Rabbit anti-His (D3I1O) monoclonal antibodies (CST, #12698, Western 1:1000, immunofluorescence 1:200); Rabbit anti-TOM20 (D8T4N) monoclonal antibodies (CST, #42406, Western 1:1000, immunofluorescence 1:200); Rabbit anti-p62 (D6M5X) monoclonal antibodies (CST, #23214, immunofluorescence 1:200); Mouse anti-FLAG (M2) monoclonal antibodies (Sigma, #F1804, Western 1:1000); Rabbit anti-TIM23 polyclonal antibodies (Proteintech, #11123-1-AP, Western 1:1000); Rabbit anti-p62 polyclonal antibodies (Proteintech, #18420-1-AP, Western 1:1000); Rabbit anti-p62 (PS00-61) monoclonal antibodies (Huabio, #HA721171, Western 1:1000); Rabbit anti-PINK1 polyclonal antibodies (Proteintech, #23274-1-AP, Western 1:500); Mouse anti-β-Tubulin polyclonal antibodies (Proteintech, #10068-1-AP, Western 1:1000); Mouse anti-HSP60 (H-1) monoclonal antibodies (Santa Cruz, #sc-13115, immunofluorescence 1:200); Rabbit anti-RNF213 polyclonal antibodies (Merck, #HPA003347, immunofluorescence 1:100, Western 1:500); Rabbit anti-PDI polyclonal antibodies (Proteintec, #11245-1-AP, Western 1:500); Rabbit anti-GM130 polyclonal antibodies (Beyotime, #AF7005, Western 1:500); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Beyotime, #A0208, Western 1:1000); HRP-conjugated goat anti-mouse IgG (H + L) secondary antibodies (Beyotime, #A0216, Western 1:1000); Goat anti-rabbit IgG (H + L) Alexa Fluor 488 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11008, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 405 cross-adsorbed secondary antibodies (Thermo Fisher, #A-31553, immunofluorescence 1:200); Goat anti-rabbit IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11012, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11032, immunofluorescence 1:200).

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Western Blot, Membrane, Fluorescence, Sequencing, Binding Assay, Knockdown

Journal: Nature Communications

Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

doi: 10.1038/s41467-024-45961-1

Figure Lengend Snippet: a Western blots showing that knock-down of p62 in HeLa cells inhibits PorB K117Q-dependent degradation of TOM20 and TIM23. b Reduced p62 and HSP60 colocalization in HeLa cells expressing PorB K5 or K null compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10 −15 ; WT PorB-PorB K5: P < 10 −15 ; WT PorB-PorB K null : P < 10 −15 ). c Western blots of HeLa 4KO cells expressing WT or truncated p62 show that the p62 LIR domain and UBI domain are indispensable for PorB K117Q-induced degradation of TOM20 and TIM23. d Western blots showing co-immunoprecipitation of ubiquitin and p62 after immunoprecipitation of PorB WT and PorB K117Q. e Western blots showing co-immunoprecipitation of both K63-linked and K48-linked polyubiquitin, after immunoprecipitation of PorB from HeLa cells, which is enhanced with inhibitors for autolysosomal degradation (BafA1, 3-MA) for K63-linked ubiquitin or proteasomal degradation (MG132) for K48-linked ubiquitin. f Colocalization between HSP60 and K63-linked polyubiquitin, but not K48-linked polyubiquitin, is induced in HeLa cells expressing PorB. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, two-tailed Mann–Whitney test, K63 only Empty-PorB K117Q: P < 10 −15 . g Western blots after immunoprecipitation of HA-PorB from HeLa cells show co-immunoprecipitation of K63-linked polyubiquitin is dependent on PorB lysine 171 and co-immunoprecipitation of K48-linked polyubiquitin is dependent on PorB lysine 128. h Western blots showing PorB-induced degradation of TOM20 and TIM23 in HeLa cells is dependent on PorB lysines 117 and 171. i Western blots showing gonococcal OMVs expressing PorB K117Q/K171Q lost the ability to induce degradation of TOM20 and TIM23. Cells in b and f are from 3 independent experiments. Western blots in a , c , d , e , g , h , i are representative of 3 independent exper i ments. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence staining or Western analysis, the following antibodies were used: Rabbit anti-LC3A/B (D3U4C) monoclonal antibodies (CST, #12741, Western 1:1000, immunofluorescence 1:200); Rabbit anti-LAMP1 (D2D11) monoclonal antibodies (CST, #9091, immunofluorescence 1:200); Rabbit anti-HA (C29F4) monoclonal antibodies (CST, #3724, Western 1:1000); Rabbit anti-His (D3I1O) monoclonal antibodies (CST, #12698, Western 1:1000, immunofluorescence 1:200); Rabbit anti-TOM20 (D8T4N) monoclonal antibodies (CST, #42406, Western 1:1000, immunofluorescence 1:200); Rabbit anti-p62 (D6M5X) monoclonal antibodies (CST, #23214, immunofluorescence 1:200); Mouse anti-FLAG (M2) monoclonal antibodies (Sigma, #F1804, Western 1:1000); Rabbit anti-TIM23 polyclonal antibodies (Proteintech, #11123-1-AP, Western 1:1000); Rabbit anti-p62 polyclonal antibodies (Proteintech, #18420-1-AP, Western 1:1000); Rabbit anti-p62 (PS00-61) monoclonal antibodies (Huabio, #HA721171, Western 1:1000); Rabbit anti-PINK1 polyclonal antibodies (Proteintech, #23274-1-AP, Western 1:500); Mouse anti-β-Tubulin polyclonal antibodies (Proteintech, #10068-1-AP, Western 1:1000); Mouse anti-HSP60 (H-1) monoclonal antibodies (Santa Cruz, #sc-13115, immunofluorescence 1:200); Rabbit anti-RNF213 polyclonal antibodies (Merck, #HPA003347, immunofluorescence 1:100, Western 1:500); Rabbit anti-PDI polyclonal antibodies (Proteintec, #11245-1-AP, Western 1:500); Rabbit anti-GM130 polyclonal antibodies (Beyotime, #AF7005, Western 1:500); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Beyotime, #A0208, Western 1:1000); HRP-conjugated goat anti-mouse IgG (H + L) secondary antibodies (Beyotime, #A0216, Western 1:1000); Goat anti-rabbit IgG (H + L) Alexa Fluor 488 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11008, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 405 cross-adsorbed secondary antibodies (Thermo Fisher, #A-31553, immunofluorescence 1:200); Goat anti-rabbit IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11012, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11032, immunofluorescence 1:200).

Techniques: Western Blot, Knockdown, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

doi: 10.1038/s41467-024-45961-1

Figure Lengend Snippet: a MS/MS spectrum of RNF213 peptide identified by mass spectrometry analysis of proteins co-immunoprecipitated with PorB from HeLa cells. b Western blots after immunoprecipitation of PorB from HeLa cells show co-immunoprecipitation of RNF213. c HeLa cells expressing PorB show colocalization between PorB, HSP60 and RNF213. Scale bar, 5 μm. d Live HeLa cell microscopy and 3D image reconstruction shows RNF213 surrounding PorB on MitoBright-labeled mitochondria. Scale bar, 5 μm. The fluorescence colocalization profile of the line is shown. e Western blots showing PorB K171-dependent enhanced co-immunoprecipitation of ubiquitin from HeLa cells transfected with an RNF213 expression vector. f Western blots showing that transfection of HeLa cells with RNF213 siRNA abolishes PorB K171-dependent co-immunoprecipitation of ubiquitin. g Western blots showing PorB K117Q, but not PorB K171Q, enhances PorB-induced degradation of TOM20 and TIM23 in HeLa cells transfected with an RNF213 expression vector. h Western blots showing that transfection of HeLa cells with RNF213 siRNA prevents PorB-induced degradation of TOM20 and TIM23 for PorB K117Q, while degradation remains unaffected for PorB K171Q. Western blots in b, e – h are representative of 3 independent experiments. Images in c and d are representative of 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence staining or Western analysis, the following antibodies were used: Rabbit anti-LC3A/B (D3U4C) monoclonal antibodies (CST, #12741, Western 1:1000, immunofluorescence 1:200); Rabbit anti-LAMP1 (D2D11) monoclonal antibodies (CST, #9091, immunofluorescence 1:200); Rabbit anti-HA (C29F4) monoclonal antibodies (CST, #3724, Western 1:1000); Rabbit anti-His (D3I1O) monoclonal antibodies (CST, #12698, Western 1:1000, immunofluorescence 1:200); Rabbit anti-TOM20 (D8T4N) monoclonal antibodies (CST, #42406, Western 1:1000, immunofluorescence 1:200); Rabbit anti-p62 (D6M5X) monoclonal antibodies (CST, #23214, immunofluorescence 1:200); Mouse anti-FLAG (M2) monoclonal antibodies (Sigma, #F1804, Western 1:1000); Rabbit anti-TIM23 polyclonal antibodies (Proteintech, #11123-1-AP, Western 1:1000); Rabbit anti-p62 polyclonal antibodies (Proteintech, #18420-1-AP, Western 1:1000); Rabbit anti-p62 (PS00-61) monoclonal antibodies (Huabio, #HA721171, Western 1:1000); Rabbit anti-PINK1 polyclonal antibodies (Proteintech, #23274-1-AP, Western 1:500); Mouse anti-β-Tubulin polyclonal antibodies (Proteintech, #10068-1-AP, Western 1:1000); Mouse anti-HSP60 (H-1) monoclonal antibodies (Santa Cruz, #sc-13115, immunofluorescence 1:200); Rabbit anti-RNF213 polyclonal antibodies (Merck, #HPA003347, immunofluorescence 1:100, Western 1:500); Rabbit anti-PDI polyclonal antibodies (Proteintec, #11245-1-AP, Western 1:500); Rabbit anti-GM130 polyclonal antibodies (Beyotime, #AF7005, Western 1:500); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (Beyotime, #A0208, Western 1:1000); HRP-conjugated goat anti-mouse IgG (H + L) secondary antibodies (Beyotime, #A0216, Western 1:1000); Goat anti-rabbit IgG (H + L) Alexa Fluor 488 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11008, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 405 cross-adsorbed secondary antibodies (Thermo Fisher, #A-31553, immunofluorescence 1:200); Goat anti-rabbit IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11012, immunofluorescence 1:200); Goat anti-mouse IgG (H + L) Alexa Fluor 594 cross-adsorbed secondary antibodies (Thermo Fisher, #A-11032, immunofluorescence 1:200).

Techniques: Tandem Mass Spectroscopy, Mass Spectrometry, Immunoprecipitation, Western Blot, Expressing, Microscopy, Labeling, Fluorescence, Ubiquitin Proteomics, Transfection, Plasmid Preparation