rabbit anti human galc polyclonal antibody  (Proteintech)

Journal: Oncology reports

Article Title: GALC gene is downregulated by promoter hypermethylation in Epstein-Barr virus-associated nasopharyngeal carcinoma.

doi: 10.3892/or.2015.4134

Figure Lengend Snippet: Figure 2. GALC protein expression in chronic nasopharyngitis and NPC. The expression of GALC protein was detected in all the samples following immuno histochemical staining. For GALC, cytoplasmic staining was considered positive. The image shows two specimens with positive and negative staining. (A) GALC protein expression is observed in nasopharyngeal mucosal columnar epithelium (magnification, x400). (B) GALC protein expression is negative in NPC cells (magnification, x400). NPC, nasopharyngeal carcinoma.

Article Snippet: Rabbit anti-human GALC polyclonal antibody was purchased from the Proteintech Group, Inc. (Chicago, IL, uSA) (working dilution, 1:200).

Techniques: Expressing, Staining, Negative Staining

Journal: Oncology reports

Article Title: GALC gene is downregulated by promoter hypermethylation in Epstein-Barr virus-associated nasopharyngeal carcinoma.

doi: 10.3892/or.2015.4134

Figure Lengend Snippet: Figure 3. Methylation status of the GALC gene promoter region in CNE-2Z cells treated with different concentrations of 5-Aza-dC (0, 5, 10, 20, 40 and 60 µM/l) for 48 h. The methylation status of the 37 CpG sites of the GALC gene promoter region is shown. (○, unmethylated CpG site; ●, methylated CpG site). 5-Aza-dC, 5-Aza-2'-deoxycytidine.

Article Snippet: Rabbit anti-human GALC polyclonal antibody was purchased from the Proteintech Group, Inc. (Chicago, IL, uSA) (working dilution, 1:200).

Techniques: Methylation

Journal: Oncology reports

Article Title: GALC gene is downregulated by promoter hypermethylation in Epstein-Barr virus-associated nasopharyngeal carcinoma.

doi: 10.3892/or.2015.4134

Figure Lengend Snippet: Figure 4. GALC mRNA expression detected using RT-PCR analysis. The control sample and the CNE-2Z cells were treated with different concentrations of 5-Aza-dC (5, 10, 20, 40 and 60 µM/l) for 48 h, and then subjected to RT-PCR to analyze the GALC mRNA expression levels. The GALC mRNA expression levels in the treated samples (20, 40 and 60 µM/l) significantly differed from those in the control samples. Statistical analysis was performed using one-way ANOVA, followed by the significant difference (Dunnett's T3) test. Data are presented as means ± SD, n=3. *P<0.05, treated groups vs. control. #P<0.01, 5, 10, 40 and 60 µM/l vs. 20 µM/l. &P<0.05, 5 and 10 µM/l vs. 40 µM/l. 5-Aza-dC, 5-Aza-2'-deoxycytidine.

Article Snippet: Rabbit anti-human GALC polyclonal antibody was purchased from the Proteintech Group, Inc. (Chicago, IL, uSA) (working dilution, 1:200).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Oncology reports

Article Title: GALC gene is downregulated by promoter hypermethylation in Epstein-Barr virus-associated nasopharyngeal carcinoma.

doi: 10.3892/or.2015.4134

Figure Lengend Snippet: Figure 5. GALC protein expression in CNE-2Z cells was analyzed by western blotting and immunofluorescence analysis, before and after treatment with different concentrations of 5-Aza-dC (5, 10, 20, 40 and 60 µM/l) for 48 h. (A) GALC protein expression was absent in the control group but present in the cells treated with different concentrations of 5-Aza-dC. β-actin protein was used as a positive control. (B) Immunofluorescent staining for GALC (red) and DAPI (green, nuclei). The inset is a high-magnification view. Scale bars, 50 µm. 5-Aza-dC, 5-Aza-2'-deoxycytidine.

Article Snippet: Rabbit anti-human GALC polyclonal antibody was purchased from the Proteintech Group, Inc. (Chicago, IL, uSA) (working dilution, 1:200).

Techniques: Expressing, Western Blot, Immunofluorescence, Control, Positive Control, Staining

Journal: Biochimica et biophysica acta

Article Title: Molecular cloning and knockdown of galactocerebrosidase in zebrafish: new insights into the pathogenesis of Krabbe's disease.

doi: 10.1016/j.bbadis.2014.01.008

Figure Lengend Snippet: Fig. 1. GALC activity in zebrafish. A, Brain extracts (40 μg of protein) from 4 month-old zebrafish adults (zf) were evaluated for GALC activity using the GALC substrate LRh-6-GalCer in the absence or in the presence of a 10-fold molar excess of psychosine (psy) or N-acetyl-D-sphingosine (sph). In this assay, LRh-6-GalCer is converted to LRh-6-Cer and the two fluorescent compounds are separated by thin layer chromatography and visualized under an ultraviolet lamp. Brain extracts from wild-type (wt) and GALC-deficient twitcher (twi−/−) mice were used as positive and negative controls, respectively. B, Brain extracts (10 μg of protein) from zebrafish adults (zf) and from wt and twi−/−mice were assessed for GALC activity using the 4-methylumbelliferyl-β-D-galactoside substrate (1.5 mM) in the presence of 20 μM AgNO3 to inhibit β-galactosidase activity [30]. C, Subcellular fractionation on Percoll density gradient of the brain extract of zebrafish adults. GALC activity co-sediments with the lysosomal β-hexosaminidase enzyme marker (β-hex). Values are representative of 3 independent experiments. Inset, Western blot (WB) analysis of the indicated pooled fractions of the gradient (5 μg of protein) shows the presence of an immunoreactive 30 kDa lysosomal-processed enzyme fragment and of the uncleaved 80 kDa protein (arrows) in the dense lysosome-containing fractions. D, Extracts (40 μg of protein) from zebrafish embryos at different stages of development were evaluated for GALC activity using the GALC substrate LRh-6-GalCer. blk, blank reaction in the absence of embryo extract.

Article Snippet: The membranes were incubated for 30 min in PBST [PBS containing 0.05% (v/v) Tween 20] and 5% (w/v) non-fat dried skimmed milk powder (blocking buffer) and subsequently incubated with a rabbit polyclonal anti-human GALC antibody (Proteintech, catalog no. 11991-1-AP).

Techniques: Activity Assay, Thin Layer Chromatography, Fractionation, Marker, Western Blot

Journal: Biochimica et biophysica acta

Article Title: Molecular cloning and knockdown of galactocerebrosidase in zebrafish: new insights into the pathogenesis of Krabbe's disease.

doi: 10.1016/j.bbadis.2014.01.008

Figure Lengend Snippet: Fig. 2. Structural models of Danio rerio Galca and Galcb proteins obtained by similarity modeling. A, Ribbon diagram of zebrafish Galca (left) and Galcb (right) colored by domain: β-sandwich (red), TIM barrel (blue), linker (orange), and lectin domain (green). B, Structure of the active site of GALC. The predicted structures of D. rerio Galca, in green, and Galcb, in blue, were superimposed to the known crystal structure of mouse Galc (pdb: 3zr6), in red. The organization of the active site and the position of catalytic residues T109, E198, E274 and R396 are conserved between mouse and D. rerio co-orthologs. The substrate galactose, placed in the catalytic crevice, is shown in magenta. Substrate specificity for galactose- rather than glucose-containing glycolipids is conferred by amino acids W307 and T109. Numbering of residues is relative to the mouse Galc RefSeq protein (NP_032105) and differs of 16 amino acids from the sequence of mouse GALC structure [38].

Article Snippet: The membranes were incubated for 30 min in PBST [PBS containing 0.05% (v/v) Tween 20] and 5% (w/v) non-fat dried skimmed milk powder (blocking buffer) and subsequently incubated with a rabbit polyclonal anti-human GALC antibody (Proteintech, catalog no. 11991-1-AP).

Techniques: Sequencing

Journal: Biochimica et biophysica acta

Article Title: Molecular cloning and knockdown of galactocerebrosidase in zebrafish: new insights into the pathogenesis of Krabbe's disease.

doi: 10.1016/j.bbadis.2014.01.008

Figure Lengend Snippet: Fig. 3. Biochemical characterization of zebrafish Galca and Galcb enzymes in HEK 293T transfected cells. A, Total cell extracts (4.0 μg or protein) from HEK 293T cells transfected with empty expression vector (mock) or vectors harboring the zebrafish galca or galcb cDNA were analyzed by Western blotting using a polyclonal anti-GALC antibody (iGALC); α-tubulin was used for normalization. B, The same extracts were assayed for GALC activity at pH 4.0. Values are the mean ± SD of 5 independent experiments. C, Cell extracts of mock (Δ), galca (□) and galcb (○) HEK 293T transfectants were assayed for GALC activity at different pH values of incubation and results are representative of 5 independent experiments. D, Subcellular fractionation on Percoll density gradient of the cell extracts of mock (Δ), galca (□) and galcb (○) HEK 293T transfectants. Both Galca and Galcb enzymatic activities co-sediment with the lysosomal β-hex- osaminidase enzyme marker (β-hex). Values are representative of 3 independent experiments. E, HEK 293T transfectants were incubated in the absence (black bar) or in the presence (open bar) of 5 mM mannose-6-phosphate (M6P). After 48 h, cell extracts and conditioned media were assayed for GALC and β-hexosaminidase activities and released activity was cal- culated as percentage of total activity. Values are the mean ± SD of 5 independent experiments.

Article Snippet: The membranes were incubated for 30 min in PBST [PBS containing 0.05% (v/v) Tween 20] and 5% (w/v) non-fat dried skimmed milk powder (blocking buffer) and subsequently incubated with a rabbit polyclonal anti-human GALC antibody (Proteintech, catalog no. 11991-1-AP).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Activity Assay, Incubation, Fractionation, Marker

Journal: Biochimica et biophysica acta

Article Title: Molecular cloning and knockdown of galactocerebrosidase in zebrafish: new insights into the pathogenesis of Krabbe's disease.

doi: 10.1016/j.bbadis.2014.01.008

Figure Lengend Snippet: Fig. 5. galca/galcb double knockdown in zebrafish embryos. A, RT-PCR alternative splicing pattern analysis of 48 hpf double galca/galcb morphants showing the efficacy of the two exon- skipping MOs when compared to wild-type (WT) embryos. α-Tubulin serves as control. B, Total extracts from wild-type embryos, single galca, single galcb and double galca/galcb morphants were assayed for GALC activity. Negligible activity was detectable in double galca/galcb morphants. The results are the mean ± SD of 3 independent experiments. C, No GALC immunoreactive proteins (iGALC) were found when the extracts of double galca/galcb morphants (MO) were analyzed by Western blotting using a polyclonal anti-GALC antibody. α-Tubulin was used for normalization. ctrl: control murine brain extract.

Article Snippet: The membranes were incubated for 30 min in PBST [PBS containing 0.05% (v/v) Tween 20] and 5% (w/v) non-fat dried skimmed milk powder (blocking buffer) and subsequently incubated with a rabbit polyclonal anti-human GALC antibody (Proteintech, catalog no. 11991-1-AP).

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Alternative Splicing, Control, Activity Assay, Western Blot