Journal: Retrovirology

Article Title: Efficient trapping of HIV-1 envelope protein by hetero-oligomerization with an N-helix chimera

doi: 10.1186/1742-4690-2-51

Figure Lengend Snippet: A. Comparison of consensus amino acid sequences of N-helix regions from various HIV-1 clades, and the consensus sequence that was used to make pNH-YFPgpi. The letters a and d under the consensus sequence indicate the position of corresponding amino acids on a helical wheel. B. Schematic diagram of the coding sequence regions in expression plasmids pYFPgpi and pNH-YFPgpi. SP, signal peptide; YFP, yellow fluorescent protein; NH, N-helix; GPI, gpi attachment signal. C. Western blot with anti-GFP antiserum of HeLa cells transfected with pNH-YFPgpi (lane 1), pYFPgpi (lane 2), or untransfected HeLa cells (lane 3). *, aberrant YFPgpi product. One of three independent experiments with similar results is shown.

Article Snippet: The supernatant was collected and immunoprecipitated with monoclonal anti-GFP antibody (Clontech, Palo Alto, CA) overnight and protein G beads for an additional 2 hours.

Techniques: Comparison, Sequencing, Expressing, Western Blot, Transfection

Journal: Retrovirology

Article Title: Efficient trapping of HIV-1 envelope protein by hetero-oligomerization with an N-helix chimera

doi: 10.1186/1742-4690-2-51

Figure Lengend Snippet: Western blot analysis of HeLa cells transfected with an expression vector for HIV-1 Env plus either pNH-YFPgpi (lanes 1–3) or pYFPgpi as control (lanes 4–6). Total cell lysates (lanes 1, 4) or anti-GFP immunoprecipitates (lanes 2, 3, 5, 6) were treated with furin (lanes 3, 6) or mock treated (lanes 1, 2, 4, 5) and analyzed with rabbit anti-gp120 antiserum.

Article Snippet: The supernatant was collected and immunoprecipitated with monoclonal anti-GFP antibody (Clontech, Palo Alto, CA) overnight and protein G beads for an additional 2 hours.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Control

Journal: Molecular Pain

Article Title: Role of spinal cord alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in complete Freund's adjuvant-induced inflammatory pain

doi: 10.1186/1744-8069-4-67

Figure Lengend Snippet: The relative levels of GluR2 (A and B) and GluR1 (C and D) in the crude cytosolic (A and C) and plasma membrane fractions (B and D) from the ipsilateral L 4–5 dorsal horn at 2 and 24 h following saline or CFA injection . Naïve rats ( n = 4–8) were used as a control group. Data are presented as mean ± SEM. n = 4–8 rats/treatment. * p < 0.05, ** p < 0.01 vs the corresponding naïve group. E: The specificity of the fractionation procedure. Top: representative Western blots showing the expression of N -cadherin, PSD-95, and β-actin in total soluble fraction, plasma membrane fraction, and cytosolic fraction from L 4–5 dorsal horns of naïve rats ( n = 3). Bottom: statistical summary of the densitometric analysis expressed relative to the corresponding protein levels in the total soluble fraction.

Article Snippet: The blotting membrane was blocked with 3% non-fat dry milk for 1 h and incubated overnight at 4°C with rabbit anti-GluR1 (1:200; Upstate/CHEMICON, Temecula, CA), rabbit anti-GluR2 (1:500; Upstate/CHEMICON), rabbit anti- N -cadherin (1:1,000; BD Biosciences, Palo Alto, CA), mouse anti-PSD-95 (1:1,000; Upstate/CHEMICON), or monoclonal mouse anti-β-actin (1:10,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Injection, Fractionation, Western Blot, Expressing

Journal: Frontiers in Endocrinology

Article Title: Exercise intervention improves mitochondrial quality in non-alcoholic fatty liver disease zebrafish

doi: 10.3389/fendo.2023.1162485

Figure Lengend Snippet: Swimming exercise alleviates mitochondrial dysfunction in diet-induced NAFLD model zebrafish livers. (A) Western-blot of P-AMPK, AMPK, SIRT1, PGC1α, NRF1, NRF2, and TFAM. (n=6) The protein expression levels of (B) P-AMPK/AMPK, (C) SIRT1, (D) PGC1α, (E) NRF1, (F) NRF2, and (G) TFAM according to densitometry analysis. (H) Mtnd1 and mtnd6 mRNA expression. (n=6) (I) The mRNA expression levels of genes related to fatty acid oxidation. (n=6) (J) The mRNA expression levels of genes related to mitochondrial respiratory complex subunits. (n=6) *, p < 0.05, **, p < 0.01, ***, p < 0.001. Data represent the mean, and error bars represent SEM. NAFLD, non-alcoholic fatty liver disease; N, normal diet; H, high fat diet; HE, high-fat diet plus exercise; OXPHOS, oxidative phosphorylation; Mitochondrial NADH dehydrogenase 1, mtnd1; Mitochondrial NADH dehydrogenase 6, mtnd6. Acyl-CoA dehydrogenase medium chain, acadm; Carnitine palmitoyltransferase 1A, cpt1a; Peroxisome proliferator-activated receptor alpha b, pparab ); NADH:ubiquinone oxidoreductase subunit A9a, ndufa9a ; Succinate dehydrogenase complex, subunit A, sdha ; Ubiquinol-cytochrome c reductase core protein 2b, uqcrc2b ; cytochrome c oxidase subunit 4I1, cox4i1 ; ATP synthase F1 subunit beta, atp5f1b . ns, not significant.

Article Snippet: The antibodies used were as follows: rabbit anti-GAPDH antibody (1:2000; servicebio), rabbit anti-COL1A1 antibody (1:1000; Wanleibio), rabbit anti-ACTA2 antibody (1:1000; Proteintech), rabbit anti-IL-1β antibody (1:2000; Wanleibio), mouse anti-IL10 antibody (1:1500; Proteintech), rabbit anti-DRP1 antibody (1:1500; Proteintech), rabbit anti-OPA1 antibody (1:1500; Proteintech), rabbit anti-MFN2 antibody (1:1500; Proteintech), rabbit anti-P-AMPK antibody (1:2000; Cell Signaling Technology), rabbit anti-AMPK antibody (1:1500; Proteintech), rabbit anti-PGC1α antibody (1:1000; Bioss), rabbit anti-NRF1 antibody (1:1000; Proteintech), rabbit anti-NRF2 antibody (1:2000; Proteintech), rabbit anti-TFAM antibody (1:2000; Proteintech), rabbit anti-PINK1antibody (1:2000; Proteintech), rabbit anti-PARKIN antibody (1:2000; Bioss) and rabbit anti-P62 antibody (1:2000; Proteintech).

Techniques: Western Blot, Expressing, Phospho-proteomics

Journal: Scientific Reports

Article Title: The Regenerative Potential of Amniotic Fluid Stem Cell Extracellular Vesicles: Lessons Learned by Comparing Different Isolation Techniques

doi: 10.1038/s41598-018-38320-w

Figure Lengend Snippet: Comparison of AFSC-EV protein content and EV markers. ( a ) Protein quantification of AFSC-EV preparations using the Pierce Bradford assay. Data are shown as mean ± SD n = 3. No difference was found between preparations isolated with UC, ExoQuick and TEIR. AFSC-EV preparations isolated using qEV had lower protein content than those isolated using Exo-PREP (p < 0.05). ( b ) Correlation analysis between total number of particles analyzed with Nanoparticle Tracking Analysis (Fig. ) and EV protein concentration (μg/μL) in each preparation of AFSC-EVs obtained by different isolation techniques [p = 0.25, r = 0.6 (95% CI −0.56 to 0.97)]. ( c ) Expression of canonical EV markers Hsp70, CD63, Flotillin-1, and TSG101 obtained by Western blot analysis for the different isolation techniques. All AFSC-EV isolation techniques showed no evidence of residual cellular debris, as evidenced by a lack of H3K27me3 protein expression. AFSCs (parent cells) and AFSC-conditioned medium (AFSC-CM; the initial starting material from which all techniques were derived), are shown as positive controls. Representative photo from n = 3 replicate analyses.

Article Snippet: Hsp70, CD63, Flotillin-1, and TSG101 were measured from AFSC-EVs, and from 20 μg total protein from AFSC-CM, and AFSCs using ExoAb Antibody Kit for Hsp70 (System Biosciences, Palo Alto, CA; primary antibody: rabbit anti-human, 1:1,000 dilution; secondary antibody: goat anti-rabbit HRP, 1:10,000 dilution), CD63 (System Biosciences, Palo Alto, CA; primary antibody: rabbit anti-human, 1:1,000 dilution; secondary antibody: goat anti-rabbit HRP, 1:10,000 dilution), TSG101 (Santa Cruz Biotechnology, Dallas, TX; primary antibody mouse anti-rat, 1:500 dilution; secondary antibody: goat anti-mouse HRP, 1:3,000 dilution), and Flotillin-1 (BD Transduction Laboratories, San Jose, CA; primary antibody mouse anti-rat, 1:1,000 dilution; secondary antibody: goat anti-mouse HRP, 1:3,000 dilution).

Techniques: Comparison, Bradford Assay, Isolation, Protein Concentration, Expressing, Western Blot, Derivative Assay