Journal: Molecular Medicine Reports

Article Title: Understanding the role of iron/heme metabolism in the anti‑inflammatory effects of natural sulfur molecules against lipopolysaccharide‑induced inflammation

doi: 10.3892/mmr.2025.13542

Figure Lengend Snippet: Sulfur compounds inhibit LPS-induced inflammation and iron/heme metabolism. (A) Flow cytometry showing Fe2+ levels in THP-1 cells following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. Western blot analysis of (B) transferrin receptor, ferroportin, DMT1 and STEAP3; and (C) MFRN, ABCB6, ABCB10, ALAS1, HO-1, FECH and FLVCR proteins in THP-1 cells following treatment with LPS (10 ng/ml) + NTS (3 µg/ml), MSM (200 mM) or TLR-C34 (40 µM) for 48 h. ALAS1, 5′-aminolevulinate synthase 1; DMT1, divalent metal transporter 1; Fe2+, ferrous ion; FECH, ferrochelatase; FPN, ferroportin; HO-1, heme oxygenase-1; LPS, lipopolysaccharide; MFRN, mitoferrin; MSM, methylsulfonylmethane; NTS, nontoxic sulfur; TfR, transferrin receptor; TLR, Toll-like receptor.

Article Snippet: The following primary antibodies were purchased from Santa Cruz Biotechnology, Inc.: Ferrochelatase (FECH; cat. no. sc-377377), phosphorylated (p)-IκBα (cat. no. sc-8404), TLR2 (cat. no. sc-21759), TLR4 (cat. no. sc-293072), COX-1 (cat. no. sc-19998), COX-2 (cat. no. sc-19999, IKKα/β (cat. no. sc-7607), and β-actin (cat. no. sc-47778); from Cell Signaling Technology, Inc.: IL-1β (cat. no. 12703), p-ERK (cat. no. 9101), ERK (cat. no. 9102), IκBα (cat. no. 9242), p-IKKα/β (cat. no. 2697), NF-κB (cat. no. 8242), p-p38 (cat. no. 4511), p38 (cat. no. 8690), ATR (cat. no. 2790), ATM (cat. no. 2873), Chk2 (cat. no. 2662), BRCA1 (cat. no. 9010), p53 (cat. no. 9282), p-MDM2 (cat. no. 3521), MDM2 (cat. no. 86934) and the DNA Damage Antibody Sampler Kit (cat. no. 9947; containing p-ATM, p-ATR, p-Chk2, p-BRCA1 and p-p53 antibodies); from Abcam: Divalent metal transporter (DMT)1 (cat. no. ab55735), 5′-aminolevulinate synthase (ALAS)1 (cat. no. ab84962), STEAP3 (cat. no. ab151566), HO-1 (cat. no. ab137749), transferrin receptor (TfR; cat. no. ab84036), PKCα (cat. no. ab179523), p-PKCα (cat. no. ab59411), TNF-α (cat. no. ab183218), IL-6 (cat. no. ab6672) and TATA-binding protein (TBP; cat. no. ab818); from LifeSpan BioSciences, Inc.: ABCB10 (cat. no. LS-C381841) and FLVCR1 (cat. no. LS-C750126); from LS Bio; Vector Laboratories, Inc.: FPN (cat. no. NBP1-21502) and iNOS (cat. no. NB300-605); from Novus Biologicals; Bio-Techne: Mitoferrin (MFRN; cat. no. MBS6013473); and from Boster Biological Technology: ABCB6 (cat. no. PA1723).

Techniques: Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1101/2022.10.21.513191

Figure Lengend Snippet: HEK-293T cells were transfected with decreasing amounts of NanoLuc and HaloTag constructs. Transfection started at 1 μg and went down to 0.125 μg per construct. “Ctrl” stands for the technical negative control to which DMSO is added instead of ligand. Proteins pairs serving as negative controls (ABCB2-ABCD1, ABCB3-ABCD1, ABCD1-ABCB5β) ( A ) and a positive control pair (ABCB2-ABCB3) ( B ) were tested along with ABCB5β-ABCB6, ABCB5β-ABCB8, and ABCB5β-ABCB9 ( C ). Data are presented as mean ± SD. Simple linear regression was used to test if the amount transfected significantly influenced the NanoBRET ratio ( n = 3). P -values are represented on graphs as *** p <0.001, ** p <0.01, and p >0.05. For ABCB2-ABCD1 ( R = 0.8719, F (1,10) = 68.07), ABCB3-ABCD1 ( R = 0.6144, F (1,10) = 15.94), ABCB5β-ABCD1 ( R = 0.6661, F (1,10) = 19.95), and ABCB5β-ABCB8 ( R = 0.7526, F (1,10) = 30.41), the relation between both parameters was statistically significant. For ABCB2-ABCB3 ( R = 0.0269, F (1,10) = 0.2767), ABCB5β-ABCB6 ( R = 0.0011, F (1,10) = 0.0113), and ABCB5β-ABCB9 ( R = 0.0204, F (1,10) = 0.2080), changes in the amount of transfected DNA did not influence the NanoBRET ratio.

Article Snippet: Four hours later, transfection with jetPRIME (Polyplus transfection, Illkirch, France) was carried out following the manufacturer’s instructions. shRNAs constructs for stable knockdown of human ABCB6 (sc-94721-SH) and ABCB9 (sc-60115-SH) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Transfection, Construct, Negative Control, Positive Control

Journal: bioRxiv

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1101/2022.10.21.513191

Figure Lengend Snippet: (A) Concentration of donor is held constant while concentration of acceptor is increased. If the interaction is specific, the signal will reach a plateau where all the donors are saturated with acceptor, and luminescence will no longer increase. On the other hand, for a nonspecific interaction, the signal will continue to increase linearly. For each square, the schematic gray halo represents the intensity of the NanoBRET signal observed from low to high. In graphs ( B-C ), the percent of the maximum NanoBRET ratio is plotted against the NanoLuc−HaloTag ratio. ( B) Donor saturation assay for positive, ABCB2-ABCB3, and negative, ABCB2-ABCD1, ABCB3-ABCD1, ABCB5β-ABCD1, controls. ( C) Donor saturation assay for ABCB5β-ABCB6, ABCB5β-ABCB8, and ABCB5β-ABCB9.

Article Snippet: Four hours later, transfection with jetPRIME (Polyplus transfection, Illkirch, France) was carried out following the manufacturer’s instructions. shRNAs constructs for stable knockdown of human ABCB6 (sc-94721-SH) and ABCB9 (sc-60115-SH) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Concentration Assay, Saturation Assay

Journal: bioRxiv

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1101/2022.10.21.513191

Figure Lengend Snippet: Cell lysates were immunoprecipitated (IP) using an anti-ABCB5, anti-ABCB6, anti-ABCB9, or anti-mCherry antibody. The precipitated proteins were visualized by SDS-PAGE using the corresponding antibody indicated on the right side of each blot. Fifty μg of proteins and the total volume obtained after IP were loaded on the gel for the lysate or IP conditions, respectively. An isotype control was performed to determine the specificity of the signal obtained in the western blots.

Article Snippet: Four hours later, transfection with jetPRIME (Polyplus transfection, Illkirch, France) was carried out following the manufacturer’s instructions. shRNAs constructs for stable knockdown of human ABCB6 (sc-94721-SH) and ABCB9 (sc-60115-SH) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Immunoprecipitation, SDS Page, Control, Western Blot

Journal: bioRxiv

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1101/2022.10.21.513191

Figure Lengend Snippet: Mel JuSo ( A ) or UACC-257 ( B ) proteins were immunoprecipitated (IP) with an anti-ABCB5, anti-ABCB6, or anti-ABCB9 antibody. The precipitated proteins were revealed by western blotting after SDS-PAGE using the corresponding antibody indicated on the right side of each blot. Fifteen μg of proteins from the starting cell lysate were loaded in the first lane and the total IP eluate was loaded on the gel. ABCB5β was expressed in High Five insect cells, and crude membrane proteins were prepared and loaded on the gel (High5 ABCB5β) as a complementary molecular weight marker when using anti-ABCB5. An isotype control was performed to determine the specificity of the signal obtained in western blot.

Article Snippet: Four hours later, transfection with jetPRIME (Polyplus transfection, Illkirch, France) was carried out following the manufacturer’s instructions. shRNAs constructs for stable knockdown of human ABCB6 (sc-94721-SH) and ABCB9 (sc-60115-SH) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Immunoprecipitation, Western Blot, SDS Page, Membrane, Molecular Weight, Marker, Control

Journal: bioRxiv

Article Title: Identification of two novel heterodimeric ABC transporters in melanoma: ABCB5β/B6 and ABCB5β/B9

doi: 10.1101/2022.10.21.513191

Figure Lengend Snippet: (A) Confocal images of PLA in Mel JuSo and UACC 257 that stably expressed either a nontargeting short hairpin RNA (shRNA) or an ABCB6-specific (or ABCB9-specific) shRNA. The PLA signal is detected in red. Nuclei are stained in blue using DAPI. After shRNA knockdown, efficiency was determined using RT-qPCR and western blots in Mel JuSo ( B ) and UACC 257 ( C ). 18S and tubulin were used as references for RT-qPCR and the western blots, respectively (n=2). Western blot quantification to assess protein expression was performed using ImageJ. ( D) Quantification of the average number of dots per cell was determined using ImageJ ( n = 3). Scramble and shRNA quantifications are presented along with technical negative controls, including PLA conducted without one antibody (AB) out of the two used or none. Data are presented as mean ± SD . In Mel JuSo: ABCB6 scramble (39.89 ± 14.21), ABCB6 shRNA (0.28 ± 0.60), ABCB5 AB control (0.55 ± 0.91), ABCB6 AB control (0 ± 0), ABCB9 scramble (16.89 ± 4.27), ABCB9 shRNA (0.84 ± 1.04), ABCB9 AB control (0 ± 0.01), and no antibody control (0.01 ± 0.05). In UACC 257: ABCB6 scramble (42.16 ± 10.62), ABCB6 shRNA (0.97 ± 1.00), ABCB5 AB control (0.01 ± 0.02), ABCB6 AB control (0.03 ± 0.07), ABCB9 scramble (25.39 ± 6.74), ABCB9 shRNA (1.04 ± 1.16), ABCB9 AB control (0.79 ± 0.58), and no antibody control (0 ± 0). Student’s t -test was performed between the scramble and shRNA conditions. Statistical significances are presented on graph as **** p <0.0001. ABCB5β/B6 in Mel JuSo ( t = 9.648, df = 22), ABCB5β/B9 in Mel JuSo ( t = 12,66, df = 22), ABCB5β/B6 in UACC 257 ( t = 13,38, df = 22), ABCB5β/B9 in UACC257 ( t = 12,34, df = 22).

Article Snippet: Four hours later, transfection with jetPRIME (Polyplus transfection, Illkirch, France) was carried out following the manufacturer’s instructions. shRNAs constructs for stable knockdown of human ABCB6 (sc-94721-SH) and ABCB9 (sc-60115-SH) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Stable Transfection, shRNA, Staining, Knockdown, Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: Pflugers Archiv

Article Title: Nitric oxide differentially regulates renal ATP-binding cassette transporters during endotoxemia

doi: 10.1007/s00424-007-0210-x

Figure Lengend Snippet: Gene expression of efflux transporters during endotoxemia

Article Snippet: Abcb6 , Abcb 6 , Rn00589801_m1 , 5.5 ± 0.35 , 5.9 ± 0.26 , 6.4 ± 0.25 , 6.5 ± 0.52 , 5.9 ± 0.38 , 5.4 ± 0.14 , 6.4 ± 0.25 , 6.2 ± 0.32.

Techniques: Gene Expression, Full Display Name

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-{beta}.

doi: 10.1096/fj.10-162107

Figure Lengend Snippet: Figure 4. Expression of the 3GalT4 gene in TGF--, CoCl2-, and hypoxia-treated cells. RNAs were prepared from control, TGF-, CoCl2-, or hypoxia-treated cells, as described in Mate- rials and Methods. cDNA was prepared using SuperScript III First-Strand Super Mix and digested with DNase, and quanti- tative real-time PCR was performed as described in Materials and Methods. Data were analyzed using the 2 Ct method as described previously (44) and represent fold change in gene expression relative to the nontreated control. *P 0.05; **P 0.01.

Article Snippet: Other antibodies and products were obtained as follows: GD1a-1 and anti-GD1a (from Dr. Ronald L. Schnaar, Departments of Pharmacology and Neurology, The Johns Hopkins University, Baltimore, MD, USA) (32); mouse anti-Ecad IgG1 and mouse anti- -catenin IgG1 (BD Biosciences, San Jose, CA, USA); mouse anti-N-cadherin (Ncad) IgG1 and rabbit anti-desmoplakin IgG, protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FN mAb IgG1 (Calbiochem/Merck KGaA, Darmstadt, Germany); mouse anti-HIF-2 mAb IgG1 and mouse anti-HIF-1 mAb IgG2b (Novus Biologicals, Littleton, CO, USA); mouse anti-occludin IgG1 (Invitrogen, Carlsbad, CA, USA); anti-ZONAB (ZO-1 associated nucleic acid-binding protein) and rabbit antiserum (Upstate Chemicon/Millipore Corp., Billerica, MA, USA); mouse anti-vimentin IgM and anti- -tubulin IgG1 (SigmaAldrich, St. Louis, MO, USA); rabbit anti- 3GalT4 IgG (ProteinTech, Chicago, IL, USA); Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA); horseradish peroxidase (HRP)-labeled goat anti-mouse IgG and HRP-labeled goat anti-mouse IgM (Southern Biotech, Birmingham, AL, USA); and FITC-labeled goat F(ab )2 anti-mouse IgM IgG (BioSource, Camarillo, CA, USA).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Gene Expression

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-{beta}.

doi: 10.1096/fj.10-162107

Figure Lengend Snippet: Figure 5. Inhibition of CoCl2-induced EMT by overexpressed 3GalT4 or exogenous Gg4. To study the effect of ganglioside GM1 or Gg4 on the CoCl2-induced EMT process, cells were preincubated with 50 M GM1 or Gg4 for 24 h and then were cultured in medium with 100 M CoCl2 in the continued presence of 50 M Gg4 or GM2 for 24 h. For study of the effect of overexpressed 3GalT4 on the CoCl2-induced EMT process, cells were treated with CoCl2 for 24 h and then were transfected with mouse 3GalT4 gene/pIRES-puro3 or vector only, as described in Materials and Methods. These cells were used for analysis of Gg4 and 3GalT4 expression, EMT marker molecules, and cell motility. Trans., transfection. A) For Gg4 analysis, cells (4 105/dish in a 6-cm dish) were treated with CoCl2 and transfected. GSLs were extracted and analyzed by HPTLC: a) orcinol/sulfuric acid spray; b) stained with TKH7 for Gg4. Experiments were performed in triplicate; representative HPTLC-immunostaining results are shown. c) Gg4 expression detected with TKH7 was calculated based on density analysis from 3 experiments; relative expression is shown as percentage of the control value. **P 0.01. B) For 3GalT4 expression analysis, cells (5 104/well in a 12-well plate) were treated and transfected as described above. Cells were harvested, lysed in RIPA buffer, and subjected (10 g of protein/slot) to SDS-PAGE. 3GalT4 expression in transfected cells was determined by Western blot as described in Materials and Methods. Experiments were performed in triplicate; representative Western blot results are shown (bottom). Normalized values with each loading control are shown as mean sd relative intensity on the ordinate (top). C) For the phagokinetic cell motility assay, NMuMG cells (5 104 cells/well in a 12-well plate) were treated as described above. Cells were detached with trypsin/EDTA, and 5 103 cells in complete culture medium were added onto each gold sol-coated well and incubated for 8 h, as described in Materials and Methods. Photos of track areas of 30 cells were taken. Cleared areas on gold sol were measured; values are means sd (squared pixels) from the Scion Image program. *P 0.05; ***P 0.001. D) For EMT marker protein analysis, cells were treated, and SDS-PAGE and Western blot were performed as described above. Experiments were performed in triplicate, and representative Western blot results are shown (top). Normalized values with each loading control are shown as mean sd relative intensity on the ordinate (bottom). *P 0.01–0.05; **P 0.001–0.005; NS, not significant.

Article Snippet: Other antibodies and products were obtained as follows: GD1a-1 and anti-GD1a (from Dr. Ronald L. Schnaar, Departments of Pharmacology and Neurology, The Johns Hopkins University, Baltimore, MD, USA) (32); mouse anti-Ecad IgG1 and mouse anti- -catenin IgG1 (BD Biosciences, San Jose, CA, USA); mouse anti-N-cadherin (Ncad) IgG1 and rabbit anti-desmoplakin IgG, protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FN mAb IgG1 (Calbiochem/Merck KGaA, Darmstadt, Germany); mouse anti-HIF-2 mAb IgG1 and mouse anti-HIF-1 mAb IgG2b (Novus Biologicals, Littleton, CO, USA); mouse anti-occludin IgG1 (Invitrogen, Carlsbad, CA, USA); anti-ZONAB (ZO-1 associated nucleic acid-binding protein) and rabbit antiserum (Upstate Chemicon/Millipore Corp., Billerica, MA, USA); mouse anti-vimentin IgM and anti- -tubulin IgG1 (SigmaAldrich, St. Louis, MO, USA); rabbit anti- 3GalT4 IgG (ProteinTech, Chicago, IL, USA); Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA); horseradish peroxidase (HRP)-labeled goat anti-mouse IgG and HRP-labeled goat anti-mouse IgM (Southern Biotech, Birmingham, AL, USA); and FITC-labeled goat F(ab )2 anti-mouse IgM IgG (BioSource, Camarillo, CA, USA).

Techniques: Inhibition, Cell Culture, Transfection, Plasmid Preparation, Expressing, Marker, High Performance Thin Layer Chromatography, Staining, Immunostaining, Control, SDS Page, Western Blot, Motility Assay, Incubation

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-{beta}.

doi: 10.1096/fj.10-162107

Figure Lengend Snippet: Figure 6. Inhibition of hypoxia-induced EMT by overexpressed 3GalT4 or exogenous Gg4. Cells were grown and treated with the hypoxia condition as described in Materials and Methods. GM1 or Gg4 was added, and transfection procedures were performed as described for Fig. 5. Expression of Gg4 (A) and 3GalT4 (B) in transfected cells, cell motility (C), and expression of EMT marker protein (D) affected by overexpressed 3GalT4 or exogenous Gg4 are shown as described for Fig. 5. Trans., transfection. *P 0.01–0.05; **P 0.001–0.005; ***P 0.001; NS, not significant.

Article Snippet: Other antibodies and products were obtained as follows: GD1a-1 and anti-GD1a (from Dr. Ronald L. Schnaar, Departments of Pharmacology and Neurology, The Johns Hopkins University, Baltimore, MD, USA) (32); mouse anti-Ecad IgG1 and mouse anti- -catenin IgG1 (BD Biosciences, San Jose, CA, USA); mouse anti-N-cadherin (Ncad) IgG1 and rabbit anti-desmoplakin IgG, protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FN mAb IgG1 (Calbiochem/Merck KGaA, Darmstadt, Germany); mouse anti-HIF-2 mAb IgG1 and mouse anti-HIF-1 mAb IgG2b (Novus Biologicals, Littleton, CO, USA); mouse anti-occludin IgG1 (Invitrogen, Carlsbad, CA, USA); anti-ZONAB (ZO-1 associated nucleic acid-binding protein) and rabbit antiserum (Upstate Chemicon/Millipore Corp., Billerica, MA, USA); mouse anti-vimentin IgM and anti- -tubulin IgG1 (SigmaAldrich, St. Louis, MO, USA); rabbit anti- 3GalT4 IgG (ProteinTech, Chicago, IL, USA); Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA); horseradish peroxidase (HRP)-labeled goat anti-mouse IgG and HRP-labeled goat anti-mouse IgM (Southern Biotech, Birmingham, AL, USA); and FITC-labeled goat F(ab )2 anti-mouse IgM IgG (BioSource, Camarillo, CA, USA).

Techniques: Inhibition, Transfection, Expressing, Marker

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-{beta}.

doi: 10.1096/fj.10-162107

Figure Lengend Snippet: Figure 7. Inhibition of TGF--induced EMT by overexpressed 3GalT4. Cells were cultured in culture medium overnight; then medium was replaced with fresh medium alone (control) or with fresh medium containing 1 ng/ml TGF- for 18 h. Transfection was performed as described in Materials and Methods and for Fig. 5. Cells were analyzed for Gg4 expression (A), 3GalT4 expression (B), cell motility (C), and EMT marker molecules (D), as described for Fig. 5. Trans., transfection. A) For Gg4 analysis, cells (4 105/dish in a 6-cm dish) were treated with TGF- and transfected. GSLs were extracted, and analyzed by HPTLC: a) orcinol/sulfuric acid spray; b) stained with TKH7 for Gg4. Experi- ments were performed in triplicate, and representative HPTLC immunostaining results are shown. c) Gg4 expression detected with TKH7 was calculated based on density analysis from 3 experiments and is shown as relative expression, as described for Fig. 5. **P 0.01. B) For 3GalT4 expression analysis, cells (5 104/well in a 12-well plate) were treated and transfected as described above. Cells were harvested, lysed in RIPA buffer, and subjected (10 g of protein/slot) to SDS-PAGE. 3GalT4 expression in transfected cells was determined by Western blot as described in Materials and Methods. Experiments were per- formed in triplicate; representative Western blot results are shown (bottom). Normalized values with each loading control are shown as mean sd relative intensity on the ordinate (top). *P 0.01–0.05. C) NMuMG cells were treated as described above. Phagokinetic cell motility assay was performed as described for Fig. 5. Values are means sd (squared pixels) from Scion Image. ***P 0.001. D) For EMT marker protein analysis, cells were treated as described above. Experiments were performed in triplicate. Representative Western blot results are shown (top); normalized values with each loading control are shown as mean sd relative intensity on the ordinate (bottom). *P 0.01–0.05; **P 0.001–0.005; ***P 0.001; NS, not significant.

Article Snippet: Other antibodies and products were obtained as follows: GD1a-1 and anti-GD1a (from Dr. Ronald L. Schnaar, Departments of Pharmacology and Neurology, The Johns Hopkins University, Baltimore, MD, USA) (32); mouse anti-Ecad IgG1 and mouse anti- -catenin IgG1 (BD Biosciences, San Jose, CA, USA); mouse anti-N-cadherin (Ncad) IgG1 and rabbit anti-desmoplakin IgG, protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FN mAb IgG1 (Calbiochem/Merck KGaA, Darmstadt, Germany); mouse anti-HIF-2 mAb IgG1 and mouse anti-HIF-1 mAb IgG2b (Novus Biologicals, Littleton, CO, USA); mouse anti-occludin IgG1 (Invitrogen, Carlsbad, CA, USA); anti-ZONAB (ZO-1 associated nucleic acid-binding protein) and rabbit antiserum (Upstate Chemicon/Millipore Corp., Billerica, MA, USA); mouse anti-vimentin IgM and anti- -tubulin IgG1 (SigmaAldrich, St. Louis, MO, USA); rabbit anti- 3GalT4 IgG (ProteinTech, Chicago, IL, USA); Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA); horseradish peroxidase (HRP)-labeled goat anti-mouse IgG and HRP-labeled goat anti-mouse IgM (Southern Biotech, Birmingham, AL, USA); and FITC-labeled goat F(ab )2 anti-mouse IgM IgG (BioSource, Camarillo, CA, USA).

Techniques: Inhibition, Cell Culture, Control, Transfection, Expressing, Marker, High Performance Thin Layer Chromatography, Staining, Immunostaining, SDS Page, Western Blot, Motility Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-{beta}.

doi: 10.1096/fj.10-162107

Figure Lengend Snippet: Figure 8. Expression of Ecad, -catenin, 3GalT4, and Gg4 at various times after TGF- treatment. Cells were treated with 2 ng/ml TGF- for various periods of time as indicated. A) Cells were harvested, and cell lysates prepared using RIPA buffer were subjected to Western blot analysis with antibodies against Ecad, -catenin, 3GalT4, or -tubulin. B) Cells treated as above were harvested and extracted with isopropa- nol/hexane/water (55:25:20) for Gg4 detection with mAb TKH7. as described in Materials and Methods.

Article Snippet: Other antibodies and products were obtained as follows: GD1a-1 and anti-GD1a (from Dr. Ronald L. Schnaar, Departments of Pharmacology and Neurology, The Johns Hopkins University, Baltimore, MD, USA) (32); mouse anti-Ecad IgG1 and mouse anti- -catenin IgG1 (BD Biosciences, San Jose, CA, USA); mouse anti-N-cadherin (Ncad) IgG1 and rabbit anti-desmoplakin IgG, protein A/G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA); mouse anti-FN mAb IgG1 (Calbiochem/Merck KGaA, Darmstadt, Germany); mouse anti-HIF-2 mAb IgG1 and mouse anti-HIF-1 mAb IgG2b (Novus Biologicals, Littleton, CO, USA); mouse anti-occludin IgG1 (Invitrogen, Carlsbad, CA, USA); anti-ZONAB (ZO-1 associated nucleic acid-binding protein) and rabbit antiserum (Upstate Chemicon/Millipore Corp., Billerica, MA, USA); mouse anti-vimentin IgM and anti- -tubulin IgG1 (SigmaAldrich, St. Louis, MO, USA); rabbit anti- 3GalT4 IgG (ProteinTech, Chicago, IL, USA); Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA); horseradish peroxidase (HRP)-labeled goat anti-mouse IgG and HRP-labeled goat anti-mouse IgM (Southern Biotech, Birmingham, AL, USA); and FITC-labeled goat F(ab )2 anti-mouse IgM IgG (BioSource, Camarillo, CA, USA).

Techniques: Expressing, Western Blot