human primate bcma tnfrsf17 biotinylated goat polyclonal antibody pab  (R&D Systems)

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of siRNA transfection of iMG. Cells were transfected 24 hours after iMG plating. After an additional 24 hours, cells were incubated with 2 μg/mL of pHrodo-labeled oligomerized amyloid (1-42) and imaged over a 20-hour window. (B) Confocal fluorescence images of iMG assessed for internalization of pHrodo-oligomerized amyloid beta treated as in (A). Digital phase contrast was used to visualize iMG. Images are stitched from four 20X images. Scale bar represents 400 μm. (C) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG per hour. Data are normalized to mock transfected control. (D) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG at endpoint. Data are normalized to mock transfected control. (E) RNA isolated from iMG 24 hours after transfection was used to quantify the percent CD33 mRNA expression by qPCR. (F) Change in TREM2 secretion in conditioned media from iMG was quantified 24 hours after transfection by MSD. (G) Protein lysates isolated from iMG 24 hours after transfection were used to quantify percent SYK phosphorylation by AlphaLISA. Data represent mean ± SEM using one-way ANOVA with uncorrected Fisher’s LSD (D) and Dunnett’s multiple comparison test (E-G). Data points (n-numbers) are plotted on each bar graph and scatter plot each representing an independent experimental replicate. Line plots are representative of three independent experimental replicates (C).

Article Snippet: Briefly, streptavidin-coated small spot MSD plates (Meso Scale Diagnostics, Rockville, MD) were coated with Biotinylated goat anti-human TREM2 polyclonal antibody (R&D Systems) used as a capture antibody for 1.5 hours with agitation at 600 rpm.

Techniques: Transfection, Incubation, Labeling, Fluorescence, Control, Isolation, Expressing, Phospho-proteomics, Comparison

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of AAV6-mediated transduction of iMG. Cells were transduced with an MOI of 125000 at the time of plating. On day 3, cells were incubated with 2 μg/mL of pHrodo-labeled oligomerized amyloid (1-42) and imaged over a 20-hour window. (B) Protein lysates isolated from iMG 72 hours after transduction were used to quantify percent CD33 protein expression by MSD. Data are normalized to no AAV6 transduction control. (C) Confocal fluorescence images of iMG assessed for internalization of pHrodo-oligomerized amyloid beta treated as in (A). Digital phase contrast was used to visualize iMG. Images are stitched from four 20X images. Scale bar represents 400 μm. (D) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry per hour. Data are normalized to no AAV6 transduction control. (E) Normalized intensity of internalized pHrodo-oligomerized amyloid beta in iMG transduced with or without AAV6-CD33 or AAV6-mCherry at endpoint. Data are normalized to no AAV6 transduction control. (F) TREM2 secretion in conditioned media was quantified from iMG 72 hours after transduction by MSD. (G)(left) Percent change in TREM2 secretion in conditioned media was quantified from HMC3 cells 48 hours after transduction with increasing AAV6-CD33 concentrations by MSD. Data are represented as a percent change relative to dose-matched AAV6-mCherry. (right) TREM2 secretion in conditioned media was quantified from HMC3 cells 48 hours after transduction of AAV6-CD33 by MSD. (MOI = 250000). (H) Protein lysates isolated from HMC3 cells 48 hours after transduction were used to quantify percent full length TREM2 protein expression by MSD. (MOI = 250000). Data represent mean ± SEM using one-way ANOVA with post-hoc Tukey’s multiple comparisons test (B), with uncorrected Fisher’s LSD (E), with post-hoc Dunnett multiple comparisons test (F, G) and one-tailed unpaired t-test (H). Data points (n-numbers) are plotted on each bar graph and scatter plot each representing an independent experimental replicate. Line plots represent three independent experimental replicates (D, G).

Article Snippet: Briefly, streptavidin-coated small spot MSD plates (Meso Scale Diagnostics, Rockville, MD) were coated with Biotinylated goat anti-human TREM2 polyclonal antibody (R&D Systems) used as a capture antibody for 1.5 hours with agitation at 600 rpm.

Techniques: Transduction, Incubation, Labeling, Isolation, Expressing, Control, Fluorescence, One-tailed Test

Journal: bioRxiv

Article Title: Increased CD33 levels tune activation and function of induced human microglial cells through inhibition of the TREM2 pathway

doi: 10.64898/2026.01.28.701050

Figure Lengend Snippet: (A) Schematic of the TREM2-CD33 pathway. CD33 inhibition acts on SYK activation, upstream of microglial activation pathways AKT, ERK, p38, and JNK. (B) Protein lysates isolated from THP-1 monocytes 48 hours after transduction were used to quantify percent SYK phosphorylation by MSD. (MOI = 31250) (C) 48 hours post-transduction, THP-1 cells (MOI = 31250) were treated with increasing concentrations of TREM2-targeting antibody agonist (0-10 μg/mL). After 5 minutes, protein lysates were isolated and used to quantify percent SYK phosphorylation by MSD. (D) 48-hours post-transduction of increasing concentrations of AAV6-CD33 (MOI = 0-31250), THP-1 cells were treated with a TREM2-targeting antibody agonist (2.5 ug/mL). After 5 minutes, protein lysates were isolated and used to quantify percent SYK phosphorylation by MSD. (E) Percent AKT and GSK3β phosphorylation was quantified from HMC3 cell lysates 48 hours after transduction of AAV6-CD33, quantified by MSD and ELISA, respectively. (F) Percent ERK and p38-MAPK phosphorylation was quantified from HMC3 cell lysates 48 hours after transduction of AAV6-CD33, quantified by ELISA. (G) Percent JNK phosphorylation was quantified from HMC3 cell lysates 48 hours after transduction of AAV6-CD33, quantified by ELISA. (H) (left to right) Percent expression of secreted IL10, IL1β, TNFα, and IL6 in conditioned media from HMC3 cells 24 hours after LPS challenge and 48 hours after transduction of AAV6-CD33 was quantified by MSD. Data represent mean ± SEM using one-tailed unpaired t-test (A, E-G), and two-way ANOVA using uncorrected Fisher’s LSD with a single pooled variance (H). Data points (n-numbers) are plotted on each bar graph and scatter plot each representing an independent experimental replicate. Line plots are representative of three and two independent experimental replicates (C, D, respectively).

Article Snippet: Briefly, streptavidin-coated small spot MSD plates (Meso Scale Diagnostics, Rockville, MD) were coated with Biotinylated goat anti-human TREM2 polyclonal antibody (R&D Systems) used as a capture antibody for 1.5 hours with agitation at 600 rpm.

Techniques: Inhibition, Activation Assay, Isolation, Transduction, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Expressing, One-tailed Test

Journal: Clinical and Experimental Immunology

Article Title: A novel fusion protein reduces kidney complement in experimental C3 glomerulopathy

doi: 10.1093/cei/uxag015

Figure Lengend Snippet: FHR5 1-9 FH 1-5 binds to C3, destabilizes C3 convertase and acts as a cofactor for factor I. Binding profiles of increasing amounts of FHR5 1-9 FH 1-5 to surface bound C3, C3b, iC3b, C3d and BSA (negative control), detected with both anti-CFHR5 (a) and anti-FH (b) antibodies. Data shown are mean values of triplicate measurements; error bars denote the standard deviation. C3a generated by convertase formation on surface-bound C3b: ELISA plates were coated with purified C3b. Surface C3 convertase was formed by addition of purified factor B (FB), factor D (FD), and properdin (P). Then increasing amounts of either FH (c) or the FHR5 1-9 FH 1-5 (d) were added followed by addition of C3 to enable C3a generation. To investigate factor I (FI) co-factor activity, FI along with increasing amounts of either FH (e) or the FHR5 1-9 FH 1-5 (f) were added prior to the convertase formation step. NC—negative control, no FB, FD, or P added. PC—positive control, no FH, or FHR5 1-9 FH 1-5 added. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test; **** P < 0.0001. Fluid phase co-factor assay products were visualized by western blotting (g). Cofactor activity was indicated by the cleavage of C3b as determined by the presence of C3 α-chain fragments (arrow). Negative controls: C3, C3b, C3b with FH, C3b with FI, and FHR5 1-9 FH 1-5 alone. Positive control: C3b incubated with FH and FI.

Article Snippet: After incubation and washing, bound FHR5 1-9 FH 1-5 was detected using goat polyclonal anti-CFHR5 antibody (R&D systems), followed by mouse anti-goat/sheep IgG-HRP (Sigma) and finally TMB substrate (BD).

Techniques: Binding Assay, Negative Control, Standard Deviation, Generated, Enzyme-linked Immunosorbent Assay, Purification, Activity Assay, Positive Control, Western Blot, Incubation