Journal: Nature communications

Article Title: TRPV1 regulates excitatory innervation of OLM neurons in the hippocampus.

doi: 10.1038/ncomms15878

Figure Lengend Snippet: Figure 8 | TRPV1-induced excitatory synaptogenesis requires calcium influx, NGF and BDNF. (a) Immunostain of rat hippocampal cultures with TRPV1 and BDNF. (b) Quantitation of BDNF signal in somas of TRPV1-expressing neurons normalized to surrounding non-TRPV1-expressing cells (n ¼ 15 images from three cultures; error ¼ s.e.m., significance determined by unpaired Student’s t-test with Welch’s correction, **Po0.01). (c) Immunostain of WT and TRPV1 knockout mouse cultures with the C-terminal TRPV1 antibody (which detects a remainins splice isoform in the TRPV1 knockouts and marks TRPV1-expressing cells—Fig. 1e,f), BDNF, and DAPI. (d) Quantitation of BDNF puncta/mm (left panel) and BDNF puncta intensity (right panel) on OLM neurons marked by the C-terminal TRPV1 antibody, in WT and TRPV1 knockout cultures, normalized to WT. (e) Immunostains of TRPV1, vGluT and MAP2 in cultures in control conditions, and in cultures treated with capsaicin in the presence of absence of 2 mM EGTA to block calcium influx, 1 mg ml 1 TrkA-Fc to scavenge NGF, or 0.4mg ml 1 TrkB-Fc to scavenge BDNF. (f) Quantitation of excitatory synapse number (vGluT puncta number) on TRPV1-expressing hippocampal neurons in the indicated conditions. Images used for quantitation were: control n ¼ 28, 1 mM cap. n ¼ 14, 2 mM EGTA n ¼ 21, 2 mM EGTA þ 1 mM cap. n ¼ 21, 1 mgml 1

Article Snippet: Recombinant Human TrkA Fc and TrkB Fc Chimera Proteins were from R & D Systems.

Techniques: Quantitation Assay, Expressing, Knock-Out, Control, Blocking Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Proteomic Profiling of IgG1 Producing CHO Cells Using LC/LC-SPS-MS 3 : The Effects of Bioprocessing Conditions on Productivity and Product Quality

doi: 10.3389/fbioe.2021.569045

Figure Lengend Snippet: Charge variant analysis (CVA) of produced anti-IL8 IgG1 using SCX-UV (A) . Shown are relative peak areas [%] of the main peak (gray) as well as acidic variants (light blue) and basic variants (dark blue). SPR analysis was used to determine binding affinity of the mAb to recombinant human FcRn (B) . Samples were analyzed in triplicates. Shown are measured dissociation constants (KD). Significance was determined using an ANOVA with a Tukey’s post-test (*** p ≤ 0.001).

Article Snippet: Recombinant human FcRn (R&D systems, Minnesota, United States) was immobilized on a CM5 sensor chip using standard amine coupling.

Techniques: Variant Assay, Produced, Binding Assay, Recombinant

Journal: bioRxiv

Article Title: R-spondins are BMP receptor antagonists in early embryonic development

doi: 10.1101/2020.09.10.287607

Figure Lengend Snippet: ( a ) BRE reporter assay in HEPG2 cells. Cells were transfected with siControl or siZNRF3/siRNF43 , and BMP4 with or without RSPO1-4 were added overnight as indicated. Normalized BRE activity upon BMP4 without RSPO2 stimulation was set to 100 %. ( b ) BRE reporter assay in HEPG2 cells upon ZNRF3 ΔR transfection, with or without overnight BMP4 and RSPO2 treatment as indicated. ( c ) BMP-reporter (vent2) assay in Xenopus laevis St.15 neurulae. Embryos were injected animally with reporter plasmids and the indicated Mo with or without rspo2 mRNA at 4-cell stage. Normalized vent2 activity of control Mo injected embryos with reporter plasmids was set to 1. ( d ) lmmunofluorescence microscopy (IF) in Xenopus laevis animal cap explants for Bmpr1a (green) and the plasma membrane (red) from embryos injected with mRNA as indicated, with a representative cell (top) and magnification (inset). Scale bar, 20 µm. For scheme, see . ( e ) Quantification of (d) . (f-g) BRE reporter assay in HEPG2 cells treated with BMP4 and RSPO2/RSPO2 ΔFU /RSPO2 ΔFU overnight as indicated. For domain structure of RSPO2 ΔFU /RSPO2 ΔFU , see Supplementary Fig. Sa . (h) IF for Bmpr1a (green) and plasma membrane (red) in animal cap explants injected as indicated, with a representative cell (top) and magnification (inset) showing the plasma membrane. Scale bar, 20 µm. For domain structure of Xenopus Rspo2 mutants, see Supplementary Fig. 8d. (i) Quantification of (h) . Data are the number of areas analyzed (e, i) or biological replicates (a, b, c, f, g) and displayed as mean ± SD. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001 from unpaired t-test. For the uncropped western blot images, see Source file .

Article Snippet: For ZNRF3-BMPR1A binding assay, plates were coated with recombinant human ZNRF3 Fc Chimera protein (R&D systems 7994-RF).

Techniques: Reporter Assay, Transfection, Activity Assay, Injection, Control, Microscopy, Clinical Proteomics, Membrane, Western Blot

Journal: bioRxiv

Article Title: R-spondins are BMP receptor antagonists in early embryonic development

doi: 10.1101/2020.09.10.287607

Figure Lengend Snippet: (a, b) In vitro binding assay between ZNRF3 and BMPR1A ECD mediated by RSPO1-3. ZNRF3-Fc protein was used as a bait, with sequential RSPO1-3 protein and BMPR1A ECD AP treatment. Bound BMPR1AEco to ZNRF3 was detected by chromogenicAP assay. (c, d) IF in H1581 cells transfected with BMPR1A-HA and ZNRF3-flag DNA upon RSPO2 and −3-HRP treatment for 3 h. RSPOs (red) were visualized with tyramid signal amplification. BMPR1A (blue) and ZNRF3 (green) were stained against HA and flag antibody. White arrowheads, colocalized BMPR1A/RSPO2; white arrows, colocalized BMPR1A/RSPO2-3/ZNRF3; yellow arrow, colocalized BMPR1A/RSPO2-3/ZNRF3 in magnified inset; Dashed lines, nucleus. Scale bar, 20 µm. (e-h) IF of colocalized BMPR1A (green)/ZNRF3 (red) in H1581 cells treated with siRNA as indicated. Nuclei were stained with Hoechst. Scale bar, 20 µm. (i) Quantification of BMPR1A colocalizing with ZNRF3 from (e-h) . (j-1) IF of BMPR1A (green) in H1581 cells treated with siRNA as indicated. (m) Quantification of cells harboring membrane localized BMPR1A from (j-1) . ( n ) Cell surface biotinylation assay in H1581 cells treated with BMPR1A-HA and siRNAas indicated. Co, control; R2, RSPO2; ZR, ZNRF3/RNF43 siRNA. After labeling surface proteins with Biotin, lysates were pulled down with streptavidin beads and subjected to Western blot analysis. Transferrin receptor (TfR), a loading control. TCL, Total cell lysate. Data shows representative result from three independent experiments. ( o ) BRE reporter assay in HEPG2 cells treated as indicated. MDC, monodansylcadaverin. ( p ) IF of colocalized BMPR1A (green)/ EEA1 (magenta) in H1581 cells treated with siRNA. White arrowheads, colocalized BMPR1A/EEA1 in magnified inset. (q) Quantification of (p) . ( r ) IF of colocalized BMPR1A (green)/ Lamp1 (red) in H1581 cells treated with siRNA as indicated. White arrowheads, colocalized BMPR1A/Lamp1 in magnified inset. ( s ) Quantification of (r) . Data for binding assays (a , b) are experimental replicates; IF (i, m, q, s) are the number of cells pooled from at least two independent experiments, and displayed as mean ± SD. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P<0.0001 from unpaired t-test.

Article Snippet: For ZNRF3-BMPR1A binding assay, plates were coated with recombinant human ZNRF3 Fc Chimera protein (R&D systems 7994-RF).

Techniques: In Vitro, Binding Assay, Transfection, Amplification, Staining, Membrane, Cell Surface Biotinylation Assay, Control, Labeling, Western Blot, Reporter Assay