Journal: Clinical and Experimental Vaccine Research

Article Title: Novel antigen panel for modern broad-spectrum recombinant rotavirus A vaccine

doi: 10.7774/cevr.2021.10.2.123

Figure Lengend Snippet: Characterization of rotavirus A (RVA) recombinant antigens. Lanes: VP8 * -[P8] (1), VP8 * -[P6] (2), VP8 * -[P4] (3), ep-875 (4), VP5 * (5). (A) Electrophoresis analysis, 8%–20% SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis), staining with Coomassie G-250. (B) Western blot membrane stained by Ponceau S. (C) Western blot analysis with commercial polyclonal antisera to 5 RVA strains: Wa-G1P[8], Ds-1 G2P[4], Ito-G3P[8], HOCHI-G4P[8], 69M-G8P[10] (Cat# MBS316568; MyBioSource Inc., San Diego, CA, USA) as primary antibodies and secondary anti-species antibodies conjugated with horseradish peroxidase. Positions of molecular weights (kDa) markers are indicated in the right.

Article Snippet: The membranes were next treated with commercial polyclonal antisera to five RVA strains: Wa-G1P[8], Ds-1 G2P[4], Ito-G3P[8], Hochi-G4P[8], 69M-G8P[10] (Cat#MBS316568; MyBioSource Inc., San Diego, CA, USA), at a 1:500 dilution and then with secondary anti-species antibodies conjugated with horseradish peroxidase (Cat#713-035-003; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) in a 1:10,000 dilution.

Techniques: Recombinant, Electrophoresis, SDS Page, Polyacrylamide Gel Electrophoresis, Staining, Western Blot, Membrane

Journal: Biophysical Journal

Article Title: SMIT1 Modifies KCNQ Channel Function and Pharmacology by Physical Interaction with the Pore

doi: 10.1016/j.bpj.2017.06.055

Figure Lengend Snippet: The KCNQ2 pore module is required for binding to SMIT1. (A) Topologies of SMIT1 (putative), KCNQ α-subunits, and KCNE (experimentally confirmed). (B–E). CHO cells were transfected with KCNQ2 fragments and/or SMIT1-FLAG, as indicated. All blots are each representative of n = 2 experiments. The immunoprecipitating (IP) antibodies are labeled FLAG, YFP, or GFP, and the immunoblot (IB) antibodies are labeled GFP or FLAG. The red “X” denotes fragments that did not bind to SMIT1, and the green check mark signifies SMIT1-binding fragments. (B) Cytosolic Q2 fragments. (Top) The N-terminal fragment KCNQ2(1–97) does not bind to SMIT1-FLAG. (Bottom) The C-terminal fragment KCNQ2 (321–852) does not bind to SMIT1-FLAG. (C) Transmembrane Q2 fragments. (Top) The N-terminus through the S4-S5 linker fragment (KCNQ2(1–224)) does not bind to SMIT1. (Bottom) Extension of the previous fragment to include the pore-forming S5-S6 region (KCNQ2(1–549)) enables binding to SMIT1-FLAG. (D) The S5–S6 pore-forming region fragment (KCNQ2(222–323)) binds to SMIT1-FLAG. (E) Summary of binding results. (F) Cartoon view from the extracellular side comparing the Kv channel and solute transporter approximate dimensions and hypothetical docking configurations.

Article Snippet: Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidine fluoride membranes for immunoblotting with the following antibodies, as noted: KCNQ2 (Santa Cruz Biotechnology, Santa Cruz, CA), DDK (Origene, Rockville, MD), FLAG (Sigma-Aldrich, St. Louis, MO), SMIT1 (Santa Cruz, CA), YFP (Santa Cruz), GFP (Santa Cruz; Rockland Immunochemicals, Pottstown, PA).

Techniques: Binding Assay, Transfection, Labeling, Western Blot

Journal:

Article Title: Adenovirus Serotype 30 Fiber Does Not Mediate Transduction via the Coxsackie-Adenovirus Receptor

doi: 10.1128/JVI.76.2.656-661.2002

Figure Lengend Snippet: Analysis of Ad5GFP and Ad5GFPf30 fibers by Western blot analysis. Purified Ad5GFP and Ad5GFPf30 were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and viral proteins were transferred to nitrocellulose membranes. Membranes were incubated with a primary antibody to the N terminus of Ad5 fiber and then with a peroxidase-conjugated goat anti-mouse secondary antibody. Membranes were developed with enhanced-chemiluminescence reagent. The gel is representative of at least three independent experiments from different viral isolates.

Article Snippet: The membrane was then washed four times for 5 min with PBS-0.1% Tween 20 and incubated with peroxidase-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) diluted 1:2,500 in PBS-0.1% Tween 20 for 1 h at RT.

Techniques: Western Blot, Purification, Polyacrylamide Gel Electrophoresis, Incubation

Journal: Cancer Research

Article Title: Selective Down-Regulation of Glioma-Associated Oncogene 2 Inhibits the Proliferation of Hepatocellular Carcinoma Cells

doi: 10.1158/0008-5472.can-06-3040

Figure Lengend Snippet: Figure 1. Expression of Shh pathway genes in HCC lines and tissues. A, one-step RT-PCR was done to detect the expression of Shh, Smo, Ptch1, Gli1, Gli2, and Gli3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level is for normalization purposes. Note that Gli2 is almost undetectable both in fetal and adult human livers. B, Gli2 expression detected by immunohistochemistry is shown in patient-matched representative samples of HCC and nontumor tissues (left) or from the livers of a murine HCC model or strain-matched wild-type mice (right). Much stronger Gli2 staining was observed in tumor compared with normal liver.

Article Snippet: Whole-cell lysates (40 Ag) were separated by PAGE, transferred to a nitrocellulose membrane, and immunoblotted with the primary antibodies against c-Myc (BD Biosciences, San Diego, CA) at 1:500, cyclin D1 (BD Biosciences) at 1:500, cyclin B1 (BD Biosciences) at 1:500, E2F1 (Santa Cruz Biotechnology) at 1:200, Foxm1 (Abcam) at 1:100, Bcl-2 (BD Biosciences) at 1:500, Ptch1 (Santa Cruz Biotechnology) at 1:200, p21 (Santa Cruz Biotechnology) at 1:200, p27 (Santa Cruz Biotechnology) at 1:200, Gli1 (Rockland, Gilbertsville, PA) at 1:4,000, Snail (Santa Cruz Biotechnology) at 1:200, poly(ADP-ribose) polymerase (PARP; BD Biosciences) at 1:500, caspase-3 (Alexis, San Diego, CA) at 1:250, and a-tubulin (Sigma) at 1:3,000 in 0.05% Tween 20-TBS (T-TBS) containing 5% skim milk overnight at 4jC followed by washing with T-TBS for 30 min.

Techniques: Expressing, One Step RT-PCR, Immunohistochemistry, Staining

Journal: Cancer Research

Article Title: Selective Down-Regulation of Glioma-Associated Oncogene 2 Inhibits the Proliferation of Hepatocellular Carcinoma Cells

doi: 10.1158/0008-5472.can-06-3040

Figure Lengend Snippet: Figure 2. Specific down-regulation of Gli2 leads to the decrease in expression of downstream target genes, the inhibition of cell proliferation, and an increase in apoptosis in HCC cells. SNU423 and Hep3B cells were plated on three sets of collagen-coated 96-well plates (5,000–10,000 per well) and transfected with increasing concentrations of Gli2-specific ASOs. Control oligo was also included to show the specificity of the Gli2 ASO. A, total RNA was isolated 36 h after transfection and Gli2 levels were determined by real-time RT-PCR. Data as percentage of untransfected cells (UTC). B, SNU423 cells were transfected with the Gli2 ASO along with the control oligo and total RNA was isolated at the indicated times. RT-PCR was done to detect the expression levels of Gli2, Gli1, and Ptch1. GAPDH level is shown for normalization purposes. C, caspase-3/7 activity was measured 30 to 36 h after transfection and is shown as fold induction over untransfected cells. D, cell proliferation was assessed 72 h after transfection and represented as percentage of untransfected cells. Columns, mean of triplicate or quadruplicate samples; bars, SD. Each study was repeated at least thrice.

Article Snippet: Whole-cell lysates (40 Ag) were separated by PAGE, transferred to a nitrocellulose membrane, and immunoblotted with the primary antibodies against c-Myc (BD Biosciences, San Diego, CA) at 1:500, cyclin D1 (BD Biosciences) at 1:500, cyclin B1 (BD Biosciences) at 1:500, E2F1 (Santa Cruz Biotechnology) at 1:200, Foxm1 (Abcam) at 1:100, Bcl-2 (BD Biosciences) at 1:500, Ptch1 (Santa Cruz Biotechnology) at 1:200, p21 (Santa Cruz Biotechnology) at 1:200, p27 (Santa Cruz Biotechnology) at 1:200, Gli1 (Rockland, Gilbertsville, PA) at 1:4,000, Snail (Santa Cruz Biotechnology) at 1:200, poly(ADP-ribose) polymerase (PARP; BD Biosciences) at 1:500, caspase-3 (Alexis, San Diego, CA) at 1:250, and a-tubulin (Sigma) at 1:3,000 in 0.05% Tween 20-TBS (T-TBS) containing 5% skim milk overnight at 4jC followed by washing with T-TBS for 30 min.

Techniques: Expressing, Inhibition, Transfection, Control, Isolation, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Activity Assay

Journal: Cancer Research

Article Title: Selective Down-Regulation of Glioma-Associated Oncogene 2 Inhibits the Proliferation of Hepatocellular Carcinoma Cells

doi: 10.1158/0008-5472.can-06-3040

Figure Lengend Snippet: Figure 3. Shh pathway is functionally active in HCC, and Gli2 is a major regulator of the pathway. SNU423 and Hep3B cells were plated in collagen-coated 12-well plates (100,000 per well), transfected first with a 6xGli-binding site luciferase vector, (Gli)6, along with the pRL-TK for 24 h. Next, the cells were transfected with 100 nmol/L of control oligo or ASO for Gli1, Gli2, or Gli3 for 16 h, and then, luciferase activity was determined. A, down-regulation of each Gli gene by the ASO was confirmed by real-time RT-PCR in SNU423 cells. B, reporter activity was normalized to Renilla luciferase and is shown as fold induction over pGL3-transfected cells. C, gel shift assay was done with the nuclear extracts from Gli ASO-transfected SNU423 cells in the presence of radiolabeled oligonucleotides containing consensus Gli-binding site. EMSA, electrophoretic mobility shift assay.

Article Snippet: Whole-cell lysates (40 Ag) were separated by PAGE, transferred to a nitrocellulose membrane, and immunoblotted with the primary antibodies against c-Myc (BD Biosciences, San Diego, CA) at 1:500, cyclin D1 (BD Biosciences) at 1:500, cyclin B1 (BD Biosciences) at 1:500, E2F1 (Santa Cruz Biotechnology) at 1:200, Foxm1 (Abcam) at 1:100, Bcl-2 (BD Biosciences) at 1:500, Ptch1 (Santa Cruz Biotechnology) at 1:200, p21 (Santa Cruz Biotechnology) at 1:200, p27 (Santa Cruz Biotechnology) at 1:200, Gli1 (Rockland, Gilbertsville, PA) at 1:4,000, Snail (Santa Cruz Biotechnology) at 1:200, poly(ADP-ribose) polymerase (PARP; BD Biosciences) at 1:500, caspase-3 (Alexis, San Diego, CA) at 1:250, and a-tubulin (Sigma) at 1:3,000 in 0.05% Tween 20-TBS (T-TBS) containing 5% skim milk overnight at 4jC followed by washing with T-TBS for 30 min.

Techniques: Transfection, Binding Assay, Luciferase, Plasmid Preparation, Control, Activity Assay, Quantitative RT-PCR, Gel Shift, Electrophoretic Mobility Shift Assay

Journal: Cancer Research

Article Title: Selective Down-Regulation of Glioma-Associated Oncogene 2 Inhibits the Proliferation of Hepatocellular Carcinoma Cells

doi: 10.1158/0008-5472.can-06-3040

Figure Lengend Snippet: Figure 5. Antiproliferative effects on HCC cells induced either by Gli2 down-regulation or KAAD-cyclopamine correlate with the changes in the expression of downstream target genes of the pathway. A, ASOs for each Gli gene or a negative control ASO (Con) were transfected into SNU423 and Hep3B cells at 100 nmol/L. KAAD-cyclopamine (CP) and its inactive analogue tomatidine (T) added to the culture at 10 Amol/L. Cells were cultured in 5% FBS medium for 48 h. Total lysates (40 Ag) were subjected to immunoblotting against c-Myc, Bcl-2, Ptch1, Gli1, p21, p27, cyclin D1, cyclin B1, E2F1, Foxm1b, and Snail antibodies. a-Tubulin level was used as a loading control. B, Hep3B cells were transfected with a Gli reporter vector along with the expression vectors for Gli1, Gli2, or Gli3 for 24 h. Reporter activity was normalized to Renilla luciferase and is shown as fold induction over pGL3. C, cells were first transfected with Gli expression vectors for 30 h and then incubated with or without KAAD-cyclopamine in 5% FBS medium for 30 h. Lysates were subjected to immunoblotting against PARP, caspase-3 (Casp-3), c-Myc, Bcl-2, and a-tubulin antibodies. Arrows, cleaved forms of PARP and caspase-3 protein. Asterisks, nonspecific (n.s.) bands.

Article Snippet: Whole-cell lysates (40 Ag) were separated by PAGE, transferred to a nitrocellulose membrane, and immunoblotted with the primary antibodies against c-Myc (BD Biosciences, San Diego, CA) at 1:500, cyclin D1 (BD Biosciences) at 1:500, cyclin B1 (BD Biosciences) at 1:500, E2F1 (Santa Cruz Biotechnology) at 1:200, Foxm1 (Abcam) at 1:100, Bcl-2 (BD Biosciences) at 1:500, Ptch1 (Santa Cruz Biotechnology) at 1:200, p21 (Santa Cruz Biotechnology) at 1:200, p27 (Santa Cruz Biotechnology) at 1:200, Gli1 (Rockland, Gilbertsville, PA) at 1:4,000, Snail (Santa Cruz Biotechnology) at 1:200, poly(ADP-ribose) polymerase (PARP; BD Biosciences) at 1:500, caspase-3 (Alexis, San Diego, CA) at 1:250, and a-tubulin (Sigma) at 1:3,000 in 0.05% Tween 20-TBS (T-TBS) containing 5% skim milk overnight at 4jC followed by washing with T-TBS for 30 min.

Techniques: Expressing, Negative Control, Transfection, Cell Culture, Western Blot, Control, Plasmid Preparation, Activity Assay, Luciferase, Incubation

Journal: Cancer Research

Article Title: Selective Down-Regulation of Glioma-Associated Oncogene 2 Inhibits the Proliferation of Hepatocellular Carcinoma Cells

doi: 10.1158/0008-5472.can-06-3040

Figure Lengend Snippet: Figure 6. Down-regulation of Gli2 inhibits the proliferation of cancer cells containing Smo mutations. A, RNA from HCT15 and DLD1 was subjected to RT-PCR for Shh, Ptch1, Smo, Gli1, Gli2, and Gli3 genes. GAPDH level is shown for normalization purposes. B, a luciferase vector with 6xGli-binding site (Gli)6 was transfected into colon cancer cells for 24 h and the Gli reporter activity is represented as fold induction over pGL3. C, colon cancer cells were either transfected with 100 nmol/L ASOs for each Gli gene or treated with 10 Amol/L of KAAD-cyclopamine or tomatidine. D, Gli2 ASO or control oligonucleotides were transfected into the cells in the presence of KAAD-cyclopamine or tomatidine. Cell proliferation was determined at 72 h and is shown as percentage of untransfected cells.

Article Snippet: Whole-cell lysates (40 Ag) were separated by PAGE, transferred to a nitrocellulose membrane, and immunoblotted with the primary antibodies against c-Myc (BD Biosciences, San Diego, CA) at 1:500, cyclin D1 (BD Biosciences) at 1:500, cyclin B1 (BD Biosciences) at 1:500, E2F1 (Santa Cruz Biotechnology) at 1:200, Foxm1 (Abcam) at 1:100, Bcl-2 (BD Biosciences) at 1:500, Ptch1 (Santa Cruz Biotechnology) at 1:200, p21 (Santa Cruz Biotechnology) at 1:200, p27 (Santa Cruz Biotechnology) at 1:200, Gli1 (Rockland, Gilbertsville, PA) at 1:4,000, Snail (Santa Cruz Biotechnology) at 1:200, poly(ADP-ribose) polymerase (PARP; BD Biosciences) at 1:500, caspase-3 (Alexis, San Diego, CA) at 1:250, and a-tubulin (Sigma) at 1:3,000 in 0.05% Tween 20-TBS (T-TBS) containing 5% skim milk overnight at 4jC followed by washing with T-TBS for 30 min.

Techniques: Reverse Transcription Polymerase Chain Reaction, Luciferase, Plasmid Preparation, Binding Assay, Transfection, Activity Assay, Control