Journal: Cell Reports Methods

Article Title: In situ microwave fixation provides an instantaneous snapshot of the brain metabolome

doi: 10.1016/j.crmeth.2023.100455

Figure Lengend Snippet:

Article Snippet: GLUT3, Rabbit , Alomone , Cat# AGT-023; RRID:AB_2756644.

Techniques:

Journal: Risk Management and Healthcare Policy

Article Title: Effects of AGT and AGTR1 Genetic Polymorphisms and Changes in Blood Pressure Over a Five-Year Follow-Up

doi: 10.2147/RMHP.S442983

Figure Lengend Snippet: The Hardy–Weinberg Equilibrium for AGT rs699 and AGTR1 rs5186 in the Study Population (P <0.05 Indicates Results are Not Consistent with the Hardy–Weinberg Equilibrium)

Article Snippet: TaqMan SNP genotyping primers and probes were designed by Applied Biosystems (rs699 assay ID: C_1985481_20 and rs5186 assay ID: C_3187716_10).

Techniques:

Journal: Risk Management and Healthcare Policy

Article Title: Effects of AGT and AGTR1 Genetic Polymorphisms and Changes in Blood Pressure Over a Five-Year Follow-Up

doi: 10.2147/RMHP.S442983

Figure Lengend Snippet: Baseline Characteristics of the Study Cohort Classified by SNPs and Genotype

Article Snippet: TaqMan SNP genotyping primers and probes were designed by Applied Biosystems (rs699 assay ID: C_1985481_20 and rs5186 assay ID: C_3187716_10).

Techniques:

Journal: Risk Management and Healthcare Policy

Article Title: Effects of AGT and AGTR1 Genetic Polymorphisms and Changes in Blood Pressure Over a Five-Year Follow-Up

doi: 10.2147/RMHP.S442983

Figure Lengend Snippet: Associations Between AGT and AGTR1 Polymorphisms and Changes in SBP and DBP at Baseline and at the 5-Year Follow-Up According to Mode of Inheritance

Article Snippet: TaqMan SNP genotyping primers and probes were designed by Applied Biosystems (rs699 assay ID: C_1985481_20 and rs5186 assay ID: C_3187716_10).

Techniques:

Journal: Risk Management and Healthcare Policy

Article Title: Effects of AGT and AGTR1 Genetic Polymorphisms and Changes in Blood Pressure Over a Five-Year Follow-Up

doi: 10.2147/RMHP.S442983

Figure Lengend Snippet: Association Between Haplotype Variations in AGT rs699 and AGTR1 rs5186 and SBP and DBP at Baseline and at the 5-Year Follow-Up

Article Snippet: TaqMan SNP genotyping primers and probes were designed by Applied Biosystems (rs699 assay ID: C_1985481_20 and rs5186 assay ID: C_3187716_10).

Techniques:

Journal: Risk Management and Healthcare Policy

Article Title: Effects of AGT and AGTR1 Genetic Polymorphisms and Changes in Blood Pressure Over a Five-Year Follow-Up

doi: 10.2147/RMHP.S442983

Figure Lengend Snippet: Odds ratios of hypertension and hypertension with metabolic syndrome depending on haplotype variations of AGT rs699 and AGTR1 rs5186.

Article Snippet: TaqMan SNP genotyping primers and probes were designed by Applied Biosystems (rs699 assay ID: C_1985481_20 and rs5186 assay ID: C_3187716_10).

Techniques:

Journal: Risk Management and Healthcare Policy

Article Title: Effects of AGT and AGTR1 Genetic Polymorphisms and Changes in Blood Pressure Over a Five-Year Follow-Up

doi: 10.2147/RMHP.S442983

Figure Lengend Snippet: A network of AGT and AGTR1 gene interactions using GeneMania, ( http://genemania.org ).

Article Snippet: TaqMan SNP genotyping primers and probes were designed by Applied Biosystems (rs699 assay ID: C_1985481_20 and rs5186 assay ID: C_3187716_10).

Techniques:

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Activation of angiogenin expression in macrophages by lipopolysaccharide via the TLR4/NF-κB pathway in colitis

doi: 10.3724/abbs.2024013

Figure Lengend Snippet: Table 1 Sequences of primers and siRNAs used in this study

Article Snippet: The membrane was blocked with 5% nonfat milk and then incubated with primary antibodies, including anti-ANG antibody (prepared in our own laboratory) and anti-ACTB antibody (#81115-1-RR; Proteintech, Chicago, USA), in TBST (Tris-buffered saline, 0.1% Tween 20) buffer at 4°C overnight.

Techniques: Negative Control

Journal: The Journal of Biological Chemistry

Article Title: HCN1 and HCN2 Proteins Are Expressed in Cochlear Hair Cells

doi: 10.1074/jbc.M112.375832

Figure Lengend Snippet: Molecular requirements for HCN amino terminus binding to protocadherin 15 CD3 determined with SPR for rat organ of Corti proteins. A, SPR for HCN1-specific (nonconserved) amino terminus sequence as follows: protocadherin 15 CD3 as ligand, HCN1-specific (nonconserved) amino terminus as analyte at 100 nm; 100 μm Ca2+ (light green), 1 mm EGTA (turquoise); buffer control (100 μm Ca2+, black). B, HCN1-specific amino terminus as analyte at 100 nm; 100 μm Ca2+ (pink, black, and turquoise, three repeats); buffer control (red). C, HCN1 full-length amino terminus as analyte at 100 nm; 100 μm Ca2+ (green), 26.5 μm Ca2+ (black), 1 mm EGTA (turquoise). D, conserved HCN1 amino-terminal sequence as analyte at 200 nm; 100 μm Ca2+. E, HCN2-specific amino terminus as analyte at 100 nm, 100 μm Ca2+ (red); HCN1-specific amino terminus at 100 nm, 100 μm Ca2+ (green). There is no binding of the HCN2-specific amino-terminal sequence to protocadherin 15 CD3. F, HCN4-specific (nonconserved) amino terminus as analyte at 100 nm, 100 μm Ca2+ (red); HCN1-specific (nonconserved) amino terminus as analyte at 100 nm, 100 μm Ca2+ (blue). There is no binding of HCN4 to protocadherin 15 CD3. A–F, three SPR determinations were performed for each condition/construct. (For KD values, see Table 1.) RU, response units.

Article Snippet: After centrifugation at 20,900 × g for 5 min at 4 °C to remove the beads (and nonspecific binding proteins), the supernatant was utilized for immunoprecipitation overnight (4 °C) with mouse anti-human filamin A (clone PM6/317, MAB1678, Chemicon), which crosses to rat sequence, rabbit anti-human HCN2 (targeting aa 147–161, UniProtKB/Swiss Prot {"type":"entrez-protein","attrs":{"text":"Q9UL51","term_id":"108935843","term_text":"Q9UL51"}} Q9UL51 , identical to rat HCN2 sequence, APC-030, Alomone), or goat anti-fascin-2 (EB08002, Everest Biotech, UK).

Techniques: Binding Assay, Sequencing, Construct

Journal: The Journal of Biological Chemistry

Article Title: HCN1 and HCN2 Proteins Are Expressed in Cochlear Hair Cells

doi: 10.1074/jbc.M112.375832

Figure Lengend Snippet: Amplification of HCN1 and HCN2 cDNA from cochlear inner and outer hair cells. A, agarose gel for HCN1 PCR indicating amplification from the cochlear inner and outer hair cell cDNA of predicted 397-bp product crossing intron (white arrows). Nucleotide sequencing of uncloned rat HCN1 amplification product from cochlear IHC indicated 100% identity to rat HCN1. Nested primers applied in PCR to the OHC 397-bp product elicited amplification of HCN1 cDNA with 100% identity to rat HCN1 nucleotide sequence. The adjacent lanes (IHC and OHC) with a second set of primers for HCN1 yielded negative results. B, agarose gel for amplification of HCN2 cDNA from rat cochlear outer hair cells (209 bp, crossing intron, black arrow) with 100% nucleotide identity to rat HCN2. S, 1-kb standards; B, water blanks.

Article Snippet: After centrifugation at 20,900 × g for 5 min at 4 °C to remove the beads (and nonspecific binding proteins), the supernatant was utilized for immunoprecipitation overnight (4 °C) with mouse anti-human filamin A (clone PM6/317, MAB1678, Chemicon), which crosses to rat sequence, rabbit anti-human HCN2 (targeting aa 147–161, UniProtKB/Swiss Prot {"type":"entrez-protein","attrs":{"text":"Q9UL51","term_id":"108935843","term_text":"Q9UL51"}} Q9UL51 , identical to rat HCN2 sequence, APC-030, Alomone), or goat anti-fascin-2 (EB08002, Everest Biotech, UK).

Techniques: Amplification, Agarose Gel Electrophoresis, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: HCN1 and HCN2 Proteins Are Expressed in Cochlear Hair Cells

doi: 10.1074/jbc.M112.375832

Figure Lengend Snippet: z-Stack confocal immunofluorescence analysis of HCN1 and HCN2 protein expression in the organ of Corti of the adult mouse. A and B, three-dimensional reconstruction of 43 1-μm confocal optical slices, indicating immunofluorescence (red) for HCN1 (Abcam 1:50) and HCN2 (green) (Alomone, 1:200) in the organ of Corti of the adult mouse. Immunofluorescence for HCN1 was localized to stereociliary arrays for outer hair cells, which in the reconstruction extended to subcuticular sites (A, short arrow). Immunofluorescence (red) for inner hair cell stereociliary regions was visible but more sparse (A, arrowhead). HCN2 immunoreactivity (green) co-localized with HCN1 in afferents beneath the inner hair cells (A and B, yellow, long arrows). Afferents beneath outer hair cells contained primarily immunofluorescence for HCN1 (A and B, red, medium arrows). The situation that was set up was potentially one of possible competition between HCN1 and HCN2 primary antibodies. Regions of intense immunoreactivity for HCN2 appeared adjacent to the outer hair cells, consistent in position to extensions of Deiters' cells (A, short arrow with dot). Scale bars for A and B, 20 μm. B, second position of three-dimensional reconstruction of z-stack confocal optical slices. The results were consistent with differential distribution of HCN1 and HCN2 in cells of the organ of Corti and differential expression at intracellular sites. C, 1-μm optical section 3 μm in from beginning position of the z-stack, illustrating HCN1 immunofluorescence (red, Santa Cruz Biotechnology) in cochlear outer hair cell stereociliary arrays, with arrows pointing to two individual inner rows of stereocilia in the stereociliary array. Scale bars for C–E, 4 μm. D, 1-μm optical section (paired with C), showing HCN2 immunofluorescence (green, Alomone) in OHC stereocilia (arrow). E, confocally determined overlap (yellow) of HCN1 (C) with HCN2 (D) in stereocilia of OHC (arrow). F, z-stack confocal imaging of HCN2 immunofluorescence (green) in cochlear outer hair cell stereocilia (short arrows) and inner hair cell stereocilia (medium arrow), 0.3-μm optical section (0.6 μm into z-stack) for HCN2 and phalloidin combination. Scale bars for F–H, 20 μm. G, rhodamine-coupled phalloidin detection of F-actin (red) in cochlear hair cell stereocilia. H, confocal overlap of HCN2 with phalloidin (yellow) in cochlear outer hair cell stereocilia (short arrows) and inner hair cell stereocilia (medium arrow). I, magnified view of HCN2 overlap with phalloidin in IHC stereocilia (arrow). Scale bar, 10 μm.

Article Snippet: After centrifugation at 20,900 × g for 5 min at 4 °C to remove the beads (and nonspecific binding proteins), the supernatant was utilized for immunoprecipitation overnight (4 °C) with mouse anti-human filamin A (clone PM6/317, MAB1678, Chemicon), which crosses to rat sequence, rabbit anti-human HCN2 (targeting aa 147–161, UniProtKB/Swiss Prot {"type":"entrez-protein","attrs":{"text":"Q9UL51","term_id":"108935843","term_text":"Q9UL51"}} Q9UL51 , identical to rat HCN2 sequence, APC-030, Alomone), or goat anti-fascin-2 (EB08002, Everest Biotech, UK).

Techniques: Immunofluorescence, Expressing, Imaging

Journal: The Journal of Biological Chemistry

Article Title: HCN1 and HCN2 Proteins Are Expressed in Cochlear Hair Cells

doi: 10.1074/jbc.M112.375832

Figure Lengend Snippet: Pre-embedding immunogold detection of HCN1 and HCN2 in hair cells of the organ of Corti in the adult rat. A and B, HCN1 immunogold (Alomone antibody) is found at apical and lateral sites on stereocilia of outer hair cells (arrows) (magnified in A1) not dissimilar in position to that reported for tip-link proteins (4). Also note that HCN1 immunogold in A is concentrated at subcuticular plate sites (short arrows) of outer hair cells (magnified in A2). Scale bars, 500 nm for A; 250 nm for A1 and A2; 100 nm for B. C, HCN1 immunogold (arrows, Alomone primary antibody) is localized in IHC to filaments extending at lateral positions, possibly cross-connecting stereocilia. Scale bar, 300 nm. Magnified view is shown in supplemental Fig. 3A. D, HCN1 immunogold (arrows, Alomone primary antibody) was found in type II afferent endings (A) on OHC (H), as was observed with confocal immunofluorescence with a different HCN1 antibody (Abcam) and different rodent (mouse). Scale bar, 200 nm. Magnified view is shown in supplemental Fig. 3B; HCN2 immunogold was found at lateral positions on taller stereocilia of OHC in the adult rat (E, arrows; magnified in E1) as well as at sites at the top of shorter stereocilia (F–I, arrows). As with HCN1, immunogold for HCN2 was also found at sites beneath the cuticular plate (E, short arrows, magnified in E2), but unlike HCN1, HCN2 was not found in type II afferent dendrites at the base of OHC (not illustrated), consistent with results from confocal microscopy. Scales bars, 500 nm for E; 250 nm for E1; 100 nm for E2, and F–I.

Article Snippet: After centrifugation at 20,900 × g for 5 min at 4 °C to remove the beads (and nonspecific binding proteins), the supernatant was utilized for immunoprecipitation overnight (4 °C) with mouse anti-human filamin A (clone PM6/317, MAB1678, Chemicon), which crosses to rat sequence, rabbit anti-human HCN2 (targeting aa 147–161, UniProtKB/Swiss Prot {"type":"entrez-protein","attrs":{"text":"Q9UL51","term_id":"108935843","term_text":"Q9UL51"}} Q9UL51 , identical to rat HCN2 sequence, APC-030, Alomone), or goat anti-fascin-2 (EB08002, Everest Biotech, UK).

Techniques: Immunofluorescence, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: HCN1 and HCN2 Proteins Are Expressed in Cochlear Hair Cells

doi: 10.1074/jbc.M112.375832

Figure Lengend Snippet: Quantitative PCR analysis of HCN isoform expression in organ of Corti of adult mouse for wild type and HCN1−/−. A, agarose gel resolution of PCR product for full-length HCN1 cDNA in organ of Corti for control (lane 2, predicted Δ = 2,363 bp) versus mutant (lane 3, predicted Δ = 2,144 bp), demonstrating full-length sequence in mutant with the in-frame deletion. B, nucleotide sequence for organ of Corti HCN1 in the HCN1−/−, demonstrating again, as for brain, contiguous mRNA sequence before and after the in-frame deletion of the pore filter and sixth trans-membrane region. Arrow shows splice location between exons 3 and 5 (exon 4 is deleted in the mutant). C, quantitative PCR for HCN isoforms in morphologically defined mouse cochlear organ of Corti subfraction (6) for HCN1−/− versus control. *** indicates p = 0.0026 for HCN1 in HCN1 mutant versus control ΔCt by unpaired, two-tailed t test. * indicates p = 0.054 for HCN2 in HCN1 mutant versus control. Five sets of experiments each with two replicates per point.

Article Snippet: After centrifugation at 20,900 × g for 5 min at 4 °C to remove the beads (and nonspecific binding proteins), the supernatant was utilized for immunoprecipitation overnight (4 °C) with mouse anti-human filamin A (clone PM6/317, MAB1678, Chemicon), which crosses to rat sequence, rabbit anti-human HCN2 (targeting aa 147–161, UniProtKB/Swiss Prot {"type":"entrez-protein","attrs":{"text":"Q9UL51","term_id":"108935843","term_text":"Q9UL51"}} Q9UL51 , identical to rat HCN2 sequence, APC-030, Alomone), or goat anti-fascin-2 (EB08002, Everest Biotech, UK).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Mutagenesis, Sequencing, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: HCN1 and HCN2 Proteins Are Expressed in Cochlear Hair Cells

doi: 10.1074/jbc.M112.375832

Figure Lengend Snippet: Immunoprecipitation by primary antibodies to filamin A, HCN2, and fascin-2. Immunoreactivity for filamin A detected by DAB (A) and immunofluorescence (B) (mouse MAB1678 raised to human filamin A crossing to rat; 1:1,000, clone PM6/317, Chemicon) was localized to stereocilia of both inner hair cells (long arrows) and outer hair cells (short arrows) in rat cochlea. Scale bars for A and B, 10 μm. C, lane 1, full-length filamin A (detected with filamin A primary antibody 1:1,000) in immunoprecipitation complex arising from the use of anti-filamin A for immunoprecipitation (1:100) from rat brain lysate. This antibody recognizes unprocessed filamin A (270–280 kDa, arrow, as well as 170-, 150-, and 120-kDa cleavage fragments (Chemicon)). Lane 2, negative control with brain lysate, without anti-filamin A immunoprecipitation + beads + primary and secondary antibodies; lane 3, standards. D, additional negative control for C. Immunoprecipitation with mouse IgG as negative control probed with antifilamin A. Lane 1, mouse IgG immunoprecipitation of brain lysate + beads, probed with filamin A primary + donkey anti-mouse secondary. Lane 2, mouse IgG (no immunoprecipitation) + beads, probed with filamin A primary + donkey anti-mouse secondary. Lane 3, denatured mouse IgG electrophoresis, probed with filamin A primary + donkey anti-mouse secondary. No protein was observed corresponding to molecular mass of filamin A (arrow). E, Western blot of HCN1 (104 kDa, arrow) in rat brain lysate (1:50, sc-19706, Santa Cruz Biotechnology), lane 1; lane 2, standards. F, immunoprecipitation by anti-filamin A of full-length HCN1. Lane 1, standards; lane 2, negative control with brain lysate without anti-filamin A immunoprecipitation + beads + primary and secondary antibodies; lane 3, HCN1 immunoprecipitated with anti-filamin A (arrow, 104 kDa), detected with goat anti-mouse HCN1 (carboxyl terminus) which crosses to rat HCN1 sequence (sc-19706 Santa Cruz Biotechnology). G, complex of filamin A and HCN1 also contained protocadherin 15 CD3. Lane 1, standards; lane 2, full-length protocadherin 15 CD3 (189 kDa, arrow) immunoprecipitated with anti-filamin A detected with custom primary antibody (1:10,000) (arrow). Goat anti-chick IgY-HRP (Santa Cruz Biotechnology) was used as the secondary antibody (1:10,000). Lane 3, negative control without anti-filamin A immunoprecipitation but with brain lysate and protein A beads and primary and secondary antibodies. H, Western blot of HCN2 in rat brain lysate. Lane 1, standards; lane 2, HCN2 detected with a rabbit polyclonal antibody (1:200, Alomone). Predicted molecular masses of 95 and 127 kDa corresponding to unglycosylated (arrow) and glycosylated HCN2, respectively. I, HCN2 is not detected in the complex immunoprecipitated by anti-filamin A. Lane 1, standards; lane 2, HCN2 bands observed in Western (H) are not detected in immunoprecipitated complex. Bands at 170–180 kDa may correspond to protocadherin 15 CD3, given that 5 of 15 aa in the epitope targeted by the HCN2 antibody are identical in protocadherin 15 CD3, and further given that protocadherin 15 CD3 is highly concentrated in the complex immunoprecipitated by anti-filamin A (G). J, anti-HCN2 immunoprecipitates HCN1 from rat brain lysate. Lane 1, standards; lane 2, anti-HCN2 primary antibody (1:33, Alomone) immunoprecipitates HCN1 unglycosylated (104 kDa, arrow) and glycosylated (120 and 130 kDa) forms detected with anti-HCN1 primary antibody (Santa Cruz Biotechnology); lane 3, negative control for immunoprecipitation with all components except anti-HCN2 antibody for immunoprecipitation (brain lysate and beads). K, anti-HCN2 immunoprecipitation of HCN1 complex does not include protocadherin 15 CD3. Lane 1, standards; lane 2, anti-protocadherin 15 CD3 primary antibody (1:7,500), goat anti-chick secondary antibody (1:5,000); lane 3, negative control without anti-HCN2 antibody for immunoprecipitation (brain lysate and beads). No bands were evident for either experimental (lane 2) or negative control (lane 3) with goat anti-chick IgY as the secondary antibody. In a second protocol, a bovine anti-chick secondary antibody (1:5,000) detected an ∼170-kDa protein in both experimental and negative control (not illustrated). L, HCN2 binds to fascin-2 by yeast two-hybrid co-transformation. Row 1, HCN2 carboxyl terminus in bait vector pGBKT7 plus fascin-2 in prey vector pGADT7; row 2, negative control with fascin-2 in prey construct plus empty bait construct; row 3, negative control with HCN2 in bait construct plus empty prey construct. The co-transformation screening was performed once, and then the desired colony was re-plated onto another selection media in triplicate, with negative controls. The same concentrations of yeast were plated in the grids for rows 1-3. M, anti-fascin-2 immunoprecipitation of HCN2. Lane 1, standards; lane 2, 95 kDa (arrow) and 127-kDa bands, corresponding to unglycosylated and glycosylated HCN2, respectively; lane 3 negative control without anti-fascin 2 antibody in immunoprecipitation (brain lysate and beads). N, HCN1 is in fascin-2 immunoprecipitation complex along with HCN2. Lane 1, standards; lane 2, unglycosylated (arrow) and glycosylated forms of HCN1 at 120 kDa. Lane 3, goat IgG immunoprecipitation negative control, with beads and all components except anti-fascin-2 for immunoprecipitation. Three or more immunoprecipitation experiments were carried out for each protein in a given complex.

Article Snippet: After centrifugation at 20,900 × g for 5 min at 4 °C to remove the beads (and nonspecific binding proteins), the supernatant was utilized for immunoprecipitation overnight (4 °C) with mouse anti-human filamin A (clone PM6/317, MAB1678, Chemicon), which crosses to rat sequence, rabbit anti-human HCN2 (targeting aa 147–161, UniProtKB/Swiss Prot {"type":"entrez-protein","attrs":{"text":"Q9UL51","term_id":"108935843","term_text":"Q9UL51"}} Q9UL51 , identical to rat HCN2 sequence, APC-030, Alomone), or goat anti-fascin-2 (EB08002, Everest Biotech, UK).

Techniques: Immunoprecipitation, Immunofluorescence, Negative Control, Electrophoresis, Western Blot, Sequencing, Transformation Assay, Plasmid Preparation, Construct, Selection

Journal: Molecular Metabolism

Article Title: Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment

doi: 10.1016/j.molmet.2021.101337

Figure Lengend Snippet: SIRT6 and SGLT2 expression in atherosclerotic plaques . ( A ) Representative confocal laser-scanning microscope images of SIRT6 and SGLT2 expression levels (red) in deparaffinized atherosclerotic plaques from non-diabetic patients, current SGLT2i users, and never SGLTi users. The von Willebrand factor (vWf, green) was used to properly localize the immunofluorescence signals in endothelial cells, while DAPI staining was used for nuclei counterstaining (blue). Scale Bar = 5 μm. (B) The ImageJ software carried out arbitrary fluorescence units (AFU) of SIRT6 and SGLT2. ∗∗p < 0.01 versus plaque specimen from patients without diabetes, #p < 0.05 never SGLT2i users. Protein expression levels of (C) SIRT6 (D) SGLT2 and (E) NF- B ( NF - κB ) in plaques from diabetic, non-diabetic, and diabetic SGLT2i-user patients. (F) Protein quantification was performed using β-Actin and α-tubulin as the internal control. SGLT2 protein expression detected by using anti-SGLT2 antibody from Cell Signaling Technology. Lane 1 = protein ladder molecular weight markers, lane 2 = non-diabetic, lane 3 = never SGLT2i users, lane 4 = SGLT2i users. The analysis of densitometric intensity was calculated with ImageJ software and expressed as arbitrary units (AU) with ∗∗p < 0.01 versus patients without diabetes, #p < 0.05 versus never SGLT2i users.

Article Snippet: SGLT1 Blocking Peptide (1:200, #BLP-GT031, alomone labs) was preincubated (2 h at room temperature) before the addition of anti-SGLT2 primary antibody to investigate the cross reactivity between SGLT1 and SGLT2 antigens.

Techniques: Expressing, Laser-Scanning Microscopy, Immunofluorescence, Staining, Software, Fluorescence, Molecular Weight

Journal: Molecular Metabolism

Article Title: Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment

doi: 10.1016/j.molmet.2021.101337

Figure Lengend Snippet: Atherosclerotic plaque phenotypes . ( A ) Immunochemistry for nitrotyrosine (X40), Tumor Necrosis Factor-alpha (TNF-α) (X40), collagen content (X40), macrophages (CD68) (X40), and Matrix metallopeptidase 9 (MMP-9) (X40), and in non-diabetic, current Sodium-Glucose co-transporter-2 inhibitor (SGLT2i)-user, and never SGLT2i-user asymptomatic plaques. Similar regions of plaque are shown. These results are typical of control, current SGLT2i-user, and never SGLT2i-user asymptomatic plaques. Negative controls were presented in Supplementary Figure 8 . (B) Nitrotyrosine, TNF-α, collagen content, CD68, and MMP-9 in current SGLT2i-user and never SGLT2i-user asymptomatic plaques (The box plots show the median, 25th and 75th percentiles, range, and extreme values). ∗P < 0.05 vs plaques from patients without diabetes. §P < 0.05 vs current SGLT2i-user plaques.

Article Snippet: SGLT1 Blocking Peptide (1:200, #BLP-GT031, alomone labs) was preincubated (2 h at room temperature) before the addition of anti-SGLT2 primary antibody to investigate the cross reactivity between SGLT1 and SGLT2 antigens.

Techniques:

Journal: Molecular Metabolism

Article Title: Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment

doi: 10.1016/j.molmet.2021.101337

Figure Lengend Snippet: Survival from MACE Cox regression analysis (adjusted for age, sex, BMI, blood pressure, heart rate, cholesterol, HDL cholesterol, LDL cholesterol, triglyceride levels, heart disease, hypertension, dyslipidemia, smoking, b-blockers, ACE inhibitors, calcium inhibitors, thiazide diuretics, and aspirin) according to diabetic status (A), SGLT2i therapy (B), and SGLT2 carotid atherosclerotic plaque content (C).

Article Snippet: SGLT1 Blocking Peptide (1:200, #BLP-GT031, alomone labs) was preincubated (2 h at room temperature) before the addition of anti-SGLT2 primary antibody to investigate the cross reactivity between SGLT1 and SGLT2 antigens.

Techniques:

Journal: Molecular Metabolism

Article Title: Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment

doi: 10.1016/j.molmet.2021.101337

Figure Lengend Snippet: SGLT2 inhibitor restored SIRT6 expression levels during hyperglycemia . (A, B) Representative confocal images of SIRT6 and SGLT2 (red), vimentin (green), and (G) their fluorescence intensity determination, performed by using ImageJ software and expressed as arbitrary fluorescence units. Scale Bar = 10 μm ∗p < 0.05 vs Ctr, ∗∗p < 0.01 vs Ctr, #p < 0.05 vs hGluc. (C–E) Representative Western blot images and analysis of SIRT6 and SGLT2 expression levels in endothelial cells pre-treated for 8 h with 5 μM SGLT2i before 48 h hGluc (25 mM) stress induction. Lane 1 = protein ladder molecular weight markers, lane 2 = Ctr, lane 3 = vehicle, lane 4 = SGLT2i, lane 5 = hGluc, lane 6 = SGLT2i+hGluc. (F–H) Western blot image and analysis of NF-kB and MMP-9 expression level in EC pre-treated for 8 h with 5 μM SGLT2i before 48 h hGluc (25 mM) stress induction. Lane 1 = protein ladder molecular weight markers, lane 2 = Ctr, lane 3 = vehicle, lane 4 = SGLT2i, lane 5 = hGluc, lane 6 = SGLT2i+hGluc. The analysis of densitometric intensity was calculated with ImageJ software and expressed as arbitrary units. α-Tubulin or GAPDH were used as internal control. ∗p < 0.05 vs Ctr, ∗∗p < 0.01 vs Ctr, #p < 0.05 vs hGluc.

Article Snippet: SGLT1 Blocking Peptide (1:200, #BLP-GT031, alomone labs) was preincubated (2 h at room temperature) before the addition of anti-SGLT2 primary antibody to investigate the cross reactivity between SGLT1 and SGLT2 antigens.

Techniques: Expressing, Fluorescence, Software, Western Blot, Molecular Weight

Journal: Molecular Metabolism

Article Title: Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment

doi: 10.1016/j.molmet.2021.101337

Figure Lengend Snippet: SIRT6 mediates both SGLT2 expression and endothelial response against hyperglycemia damages . (A) SIRT6 and (B) SGLT2 expression levels in EC after SIRT6 silencing and/or SGLT2i pre-treatment for 8 h. Lane 1 = protein ladder molecular weight markers, lane 2 = Ctr, lane 3 = scramble siRNA, lane 4 = SIRT6-siRNA, lane 5 = SGLT2i, lane 6 = SIRT6-siRNA+SGLT2i. α-Tubulin or GAPDH were used as internal control. (C) The analysis of densitometric intensity was calculated with ImageJ software and expressed as arbitrary units. ∗p < 0.05 vs Ctr, ∗∗p < 0.01 vs Ctr, ∗∗∗p < 0.001 vs Ctr, §p < 0.05 vs SIRT6-siRNA. (D, E) SGLT2 and (F, G) TNF-α protein levels assessed by Western blot analyses. Endothelial cells, after SIRT6 silencing, were subjected or not subjected to 8 h of pre-treatment with SGLT2i before starting 48 h of hGluc stress induction. Lane 1 = protein ladder molecular weight markers, lane 2 = Ctr, lane 3 = scramble siRNA, lane 4 = SIRT6-siRNA, lane 5 = SGLT2i, lane 6 = SIRT6-siRNA+SGLT2i, lane 7 = hGluc, lane 8 = SIRT6-siRNA+hGluc, lane 9 = SGLT2i+hGluc, lane 10 = SIRT6 siRNA+SGLT2i+hGluc. β-Actin or GAPDH were used as the internal control. (H) TNF-α cytokine levels in EC measured after SIRT6 silencing and SGLT2i pre-treatment before starting 48 h of hGluc incubation. ∗p < 0.05 vs Ctr, ∗∗p < 0.01 vs Ctr, #p < 0.05 vs hGluc, §p < 0.05 vs SIRT6 siRNA.

Article Snippet: SGLT1 Blocking Peptide (1:200, #BLP-GT031, alomone labs) was preincubated (2 h at room temperature) before the addition of anti-SGLT2 primary antibody to investigate the cross reactivity between SGLT1 and SGLT2 antigens.

Techniques: Expressing, Molecular Weight, Software, Western Blot, Incubation

Journal: Molecular Metabolism

Article Title: Sodium-glucose co-transporter2 expression and inflammatory activity in diabetic atherosclerotic plaques: Effects of sodium-glucose co-transporter2 inhibitor treatment

doi: 10.1016/j.molmet.2021.101337

Figure Lengend Snippet: SIRT6 gene silencing blocks the SGLT2i protective effects against hyperglycemia injury . (A) Cytotoxicity, assessed by LDH Assay Kit-WST, and (B, C) cytokine levels in endothelial cells measured after SIRT6 silencing and hGluc stress condition for 48 h, preceded or not by SGLT2i pre-treatment for 8 h. (D, E) Intracellular ROS content detected by flow cytometry analysis using DCF probe. EC after SIRT6-siRNA were subjected or not subjected to 8 h SGLT2i pre-treatment before starting 48 h of incubation with hGluc. (F) Representative images of confocal laser scanning analyses of mitochondrial ROS generation detected by MitoSOX probe and (G) mitochondrial superoxide levels detected by flow cytometry analysis. Results are expressed as median fluorescence intensity (MFI). Scale bars = 10 μm. The cytoskeleton is marked with Phalloidin 488 (green), while DAPI was used for the nuclei counterstain (blue). (H) Representative confocal images of NF- B ( NF - κB ) (red) and vimentin (green) and (I) fluorescence intensity analysis, performed using ImageJ software, expressed as arbitrary fluorescence units. (J, K) SIRT6 protein levels, assessed by Western blot analysis. Endothelial cells, after SGLT2 silencing, were subjected or not subjected to 8 h of pre-treatment with SGLT2i before starting 48 h of hGluc stress induction. Lane 1 = protein ladder molecular weight markers, lane 2 = Ctr, lane 3 = vehicle, lane 4 = SGLT2i, lane 5 = hGluc, lane 6 = SGLT2i+hGluc, lane 7 = scramble siRNA, lane 8 = SGLT2-siRNA, lane 9 = SGLT2-siRNA+hGluc, lane 10 = SGLT2-siRNA+SGLT2i+hGluc. The analysis of densitometric intensity was calculated with ImageJ software and expressed as arbitrary units (AU). α-Tubulin was used as an internal control. ∗p < 0.05 vs Ctr, ∗∗p < 0.01 vs Ctr, #p < 0.05 vs hGluc.

Article Snippet: SGLT1 Blocking Peptide (1:200, #BLP-GT031, alomone labs) was preincubated (2 h at room temperature) before the addition of anti-SGLT2 primary antibody to investigate the cross reactivity between SGLT1 and SGLT2 antigens.

Techniques: Lactate Dehydrogenase Assay, Flow Cytometry, Incubation, Fluorescence, Software, Western Blot, Molecular Weight