p plcγ1 tyr783  (Cell Signaling Technology Inc)

Journal: Microbiology Spectrum

Article Title: D-glucose uptake inhibits bovine alphaherpesvirus 1 post-binding cell process entry via inhibition of PLC-γ1 signaling in a glucose transporter 1-independent manner

doi: 10.1128/spectrum.02456-25

Figure Lengend Snippet: Glucose affects phosphorylation of PLC-γ1 at Ser1248. ( A ) MDBK cells were subjected to glucose depletion using D-glucose-free DMEM or mock depletion using high glucose DME for 12 h. The cells were then treated with D-glucose at the indicated concentrations in glucose-free DMEM for 0.5 h, after which cell lysates were prepared. The protein levels of p-PLC-γ1(Ser1248) and PLC-γ1 were detected by Western blotting. ( B ) MDBK cells were subjected to glucose depletion or mock depletion as described above. The cells were then infected with BoAHV-1 (MOI =1) for 0.5 h in the presence of D-glucose at the indicated concentrations in glucose-free DMEM. After the infection, the lysates were prepared. The protein levels of p-PLC-γ1(Ser1248) and PLC-γ1 were detected by Western blotting. β-Actin was used as a loading control and subsequent quantitative analysis. ( C ) A549 cells in 60 mm dishes were subjected to glucose depletion via culturing cells using glucose-free DMEM. Then, the cells were infected with BoAHV-1(MOI =1) for 0.5 h in the presence of D-glucose at a concentration of 25 mM in glucose-free DMEM. After the infection, the cell lysates were prepared. The protein levels of p-PLC-γ1(Ser1248) and PLC-γ1 were detected by Western blotting. The band intensity was analyzed with free software ImageJ. The results shown are the average of three independent experiments, with error bars indicating SDs. Significance was assessed with Student’s t -test (*, P < 0.05; **, P < 0.01; ns, not significant).

Article Snippet: Phospho-PLCγ1 (Ser1248) rabbit mAb (cat# 8713S), HRP-conjugated goat anti-mouse IgG (cat# 7076), and HRP-labeled goat anti-rabbit IgG (cat# 7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Western Blot, Infection, Control, Concentration Assay, Software

Journal: Microbiology Spectrum

Article Title: D-glucose uptake inhibits bovine alphaherpesvirus 1 post-binding cell process entry via inhibition of PLC-γ1 signaling in a glucose transporter 1-independent manner

doi: 10.1128/spectrum.02456-25

Figure Lengend Snippet: Schematic of putative model on the functional effects of GLUT1 and D-glucose on BoAHV-1 productive infection. GLUT1 plays an important role in the stimulation of β-catenin-dependent transcriptional effects, a potential mechanism to affect viral productive infection. D-glucose at higher concentration is capable of inhibiting virus entry partially via blocking PLC-γ1 signaling as demonstrated by reduced phosphorylation of p-PLC-γ1 at Ser1248. The graphic was created by the online program of BioGDP.com .

Article Snippet: Phospho-PLCγ1 (Ser1248) rabbit mAb (cat# 8713S), HRP-conjugated goat anti-mouse IgG (cat# 7076), and HRP-labeled goat anti-rabbit IgG (cat# 7074) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Functional Assay, Infection, Concentration Assay, Virus, Blocking Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Supported membrane assay probes PLCγ1 activity in LAT condensates

doi: 10.64898/2026.01.23.701188

Figure Lengend Snippet: (A) A schematic summarizing the lysate assay protocol. PLCγ1-mNG was generated in HEK293T cells, then flowed directly as dilute lysate over pLAT bilayers containing PIP 2 . Active PLCγ1-mNG hydrolyzed PIP 2 to DAG, which was detected using a labeled DAG sensor (PKCθ-C1b-SNAP-Alexa 647). Parts of the figure were drawn using images from Servier Medical Art Commons Attribution 3.0 Unported License ( http://smart.servier.com (accessed on 21 March 2022)). (B) An example TIRF image of the DAG sensor demonstrating the segmentation of 50 x 50 µm images for analysis. TIRF images in a time series were sectioned into 5 x 5 µm patches and analyzed as separate instances. (C) Images of a single 5 x 5 µm patch in the DAG sensor channel (Top) and PLCγ1-mNG channel (Bottom) through time, corresponding to the time axis for the curves below. (D) Curves obtained from processing the raw median fluorescence from membrane patches, representing the amount of DAG in the bilayer (red, left axis, foreground curves) and density of PLCγ1-mNG recruited (green, right axis, background curves) over 20 minutes. PLCγ1-mNG lysate (∼1:1000 dilution) with DAG sensor was injected at t = 0 min. To attain quantitative curves, raw fluorescence was first normalized to the time of injection. DAG was calibrated using the assumption that the average normalized maximum for multiple experiments must be 2% (given 2% PIP 2 bilayers). PLCγ1-mNG was calibrated by matching single-molecule counts (at high laser power) to bulk fluorescence (at low laser power). The two solid, darkly colored curves represent the median of all patches analyzed. Lightly colored curves in the background represent the results from each single patch (n = 100). Lipid composition (mol%): 94:4:2 DOPC:Ni-DGS:PIP 2 . LAT density: ∼1500 µm -2 .

Article Snippet: The membranes were incubated with either PLCγ1 (D9H10) XP rabbit mAb (Cell Signaling Technology, 1:1000 dilution), phospho-PLCγ1 (Tyr783) (D6M9S) rabbit mAb (Cell Signaling Technology, 1:1000 dilution), SLP-76 (D1R1A) rabbit mAb (Cell Signaling Technology, 1:1000 dilution), or Gads (UW40) mouse mAb conjugated with Alexa 647 (Santa Cruz Biotechnology, 1:200 dilution) overnight at 4 °C with agitation.

Techniques: Generated, Labeling, Fluorescence, Membrane, Injection

Journal: bioRxiv

Article Title: Supported membrane assay probes PLCγ1 activity in LAT condensates

doi: 10.64898/2026.01.23.701188

Figure Lengend Snippet: (A) Western blot probed for total PLCγ1 content. Cells were transfected with either a Gads expression vector (“PLCγ1-mNG Null”, left lane) or PLCγ1-mNG vector (middle and rightmost lanes). PLCγ1-mNG lysates were loaded after either 0 min (middle) or 60 min (right) at RT to determine the stability of PLCγ1-mNG against degradation. Each lane was loaded with 1 µL of lysate. (B) Relative blot intensities (background corrected) for endogenous PLCγ1 (left, grey) versus transfected PLCγ1-mNG (right, green) normalized to endogenous levels (same lane) (n=2). (C) Western blot probed for pY783 with a series of time points and different kinase conditions. Phosphorylation reactions were conducted in 10 µL volumes containing 1 µL of PLCγ1-mNG lysate each, ATP, phosphatase inhibitor, and kinase in buffer. Reactions were kept at RT for the designated time, then quenched with SDS-PAGE loading buffer. Either no kinase, his6-Hck, or BTK-KD (Kinase Domain) was used. (D) Blot intensities (background corrected) of pPLCγ1-mNG relative to non-specific pY bands.

Article Snippet: The membranes were incubated with either PLCγ1 (D9H10) XP rabbit mAb (Cell Signaling Technology, 1:1000 dilution), phospho-PLCγ1 (Tyr783) (D6M9S) rabbit mAb (Cell Signaling Technology, 1:1000 dilution), SLP-76 (D1R1A) rabbit mAb (Cell Signaling Technology, 1:1000 dilution), or Gads (UW40) mouse mAb conjugated with Alexa 647 (Santa Cruz Biotechnology, 1:200 dilution) overnight at 4 °C with agitation.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Phospho-proteomics, SDS Page

Journal: bioRxiv

Article Title: Supported membrane assay probes PLCγ1 activity in LAT condensates

doi: 10.64898/2026.01.23.701188

Figure Lengend Snippet: (A) A schematic showing greater detail of the lysate experiment at the membrane. Hck and LAT are functionalized to the SLB via Ni-histidine chelation. Hck phosphorylates LAT allowing for the recruitment of PLCγ1-mNG. PLCγ1-mNG is then subsequently phosphorylated by Hck at Y783 and is active at the membrane after structural rearrangement. (B) Relative observed enzymatic velocities ( v obs ) (black, left axis, defined as the enzymatic rate for the linear portion of the reaction trace) and PLCγ1-mNG densities (green, right axis, defined as density at the midpoint of v obs measurement) for various assay conditions, normalized to the median value for PLCγ1-WT-mNG lysate on a pLAT bilayer. Distributions of 100 membrane patches are shown as half-violin plots, with single observations shown as points and distributions represented by a shaded area. Compact box plots are overlayed over the distributions to represent the median (white circles), quartile regions (box), and extremes with outliers excluded (whiskers). Median values for v obs (m v ) and PLCγ1-mNG density (m ρ ) are expressly denoted for clarity. Lipid composition (mol%): 94:4:2 DOPC:Ni-DGS:PIP 2 . LAT densities: ∼1500 µm -2 .

Article Snippet: The membranes were incubated with either PLCγ1 (D9H10) XP rabbit mAb (Cell Signaling Technology, 1:1000 dilution), phospho-PLCγ1 (Tyr783) (D6M9S) rabbit mAb (Cell Signaling Technology, 1:1000 dilution), SLP-76 (D1R1A) rabbit mAb (Cell Signaling Technology, 1:1000 dilution), or Gads (UW40) mouse mAb conjugated with Alexa 647 (Santa Cruz Biotechnology, 1:200 dilution) overnight at 4 °C with agitation.

Techniques: Membrane

Journal: bioRxiv

Article Title: Supported membrane assay probes PLCγ1 activity in LAT condensates

doi: 10.64898/2026.01.23.701188

Figure Lengend Snippet: (A)-(C) Field-corrected images of LAT-Alexa 555 (Ax555) (gold, top), PLCγ1-mNG (green, middle), and DAG sensor (red, bottom) with 5 µM Grb2 and 2 µM SOS-PR in solution at t = 15 min. The entire FOV is 50 x 50 µm (scale bar = 10 µm) with an inset showing a 4X expansion of the center 5 x 5 µm patch (scale bar = 2.5 µm). D. A binary image mask for LAT segmented into condensed (gold) and disperse (grey) areas obtained from Gaussian filtration (σ=1) and thresholding of the LAT-Ax555 image in (A). Inset and scale bars are equivalent to those in (A). (E)-(F) Images showing the effect of applying the binary LAT mask to PLCγ1-mNG (green) and DAG sensor (red) channels. Only the outline of the applied mask is shown for visibility. Insets and scale bars are equivalent to those in (B-C). (G)-(I) Histograms of relative pixel intensities for LAT-Ax555 (gold, top), PLCγ1-mNG (green, middle), and DAG sensor (red, bottom). Pixel intensities were normalized to the median value for all pixels. Values greater than 1 indicate an increase in brightness from the overall median, while values less than 1 indicate a decrease. Histograms for all pixels are displayed in full color in the background. Histograms for pixels that are in areas of condensed LAT are shown in translucent white in the foreground. Pixel histograms for disperse LAT areas are shown in translucent grey. Dotted lines denoting the median of each histogram are decorated with an icon corresponding to the LAT mask for all pixels (gold and grey), LAT condensed (gold only), or LAT disperse (grey only). Median fold change values for each histogram are displayed in the top right for clarity. Lipid compositions for all experiments (mol%): 94:4:2 DOPC:Ni-DGS:PIP 2 . LAT density: ∼1500 µm -2 (10% labeled).

Article Snippet: The membranes were incubated with either PLCγ1 (D9H10) XP rabbit mAb (Cell Signaling Technology, 1:1000 dilution), phospho-PLCγ1 (Tyr783) (D6M9S) rabbit mAb (Cell Signaling Technology, 1:1000 dilution), SLP-76 (D1R1A) rabbit mAb (Cell Signaling Technology, 1:1000 dilution), or Gads (UW40) mouse mAb conjugated with Alexa 647 (Santa Cruz Biotechnology, 1:200 dilution) overnight at 4 °C with agitation.

Techniques: Filtration, Labeling

Journal: bioRxiv

Article Title: Supported membrane assay probes PLCγ1 activity in LAT condensates

doi: 10.64898/2026.01.23.701188

Figure Lengend Snippet: (A) TIRF images (5 x 5 µm, scale bars = 2 µm) of PLCγ1-mNG in different conditions corresponding to the distributions below. From left to right: PLCγ1-mNG only (with 2 µL control lysate), PLCγ1-mNG + Gads:SLP76 (from lysate, 1 µL each), PLCγ1-mNG + Grb2:SOS-PR (purified, 6 µM:2 µM respectively), and PLCγ1-mNG + all previously described adapters. LAT-Ax555 (10% labeled) was used in this experiment but was determined to have negligible contribution in the PLCγ1-mNG channel. (B) Half-violin plots showing distributions of relative v obs (grey, left axis) and relative PLCγ1-mNG density (green, right axis), normalized to PLCγ1 only conditions. Compact box plots overlay the distributions to convey statistics. Median values for v obs (m v ) and PLCγ1-mNG density (m PLCγ1 ) are expressly denoted for clarity. (C) TIRF images (5 x 5 µm, scale bars = 2 µm) of PLCγ1-mNG in different conditions corresponding to the distributions below. From left to right: PLCγ1-mNG only (no control lysate), PLCγ1-mNG pre-phosphorylated by 1 µM BTK-KD in solution for 1 hr, and similarly pre-phosphorylated PLCγ1-mNG + Grb2:SOS-PR (purified, 6 µM:2 µM respectively). Hck was on the membrane for all experiments. Unlabeled LAT was used in this experiment. (D) Half-violin plots showing distributions of relative v obs (grey, left axis) and relative PLCγ1-mNG density (green, right axis), normalized to PLCγ1 only conditions (no pre-phosphorylation). Compact box plots overlay the distributions to convey statistics. Median values for v obs (m v ) and PLCγ1-mNG density (m ρ ) are expressly denoted for clarity. Lipid compositions for all experiments (mol%): 94:4:2 DOPC:Ni-DGS:PIP 2 . LAT densities: ∼1500 µm -2 .

Article Snippet: The membranes were incubated with either PLCγ1 (D9H10) XP rabbit mAb (Cell Signaling Technology, 1:1000 dilution), phospho-PLCγ1 (Tyr783) (D6M9S) rabbit mAb (Cell Signaling Technology, 1:1000 dilution), SLP-76 (D1R1A) rabbit mAb (Cell Signaling Technology, 1:1000 dilution), or Gads (UW40) mouse mAb conjugated with Alexa 647 (Santa Cruz Biotechnology, 1:200 dilution) overnight at 4 °C with agitation.

Techniques: Control, Purification, Labeling, Membrane, Phospho-proteomics

Journal: bioRxiv

Article Title: Supported membrane assay probes PLCγ1 activity in LAT condensates

doi: 10.64898/2026.01.23.701188

Figure Lengend Snippet: (A) Graphic of PLCγ1 binding to pLAT and fully engaging the membrane, where full PLCγ1 activity is observed in experimental results. (B) A graphic of PLCγ1 in the LAT condensate, where PLCγ1 activity is reduced in experiments. Both Grb2 dimers and Grb2:SOS-PR interactions can crosslink LAT. (C) Graphic showing PLCγ1 interactions with Gads:SLP76 in the absence of a larger condensate. PLCγ1 is fully active in experiments with this configuration. (D) Graphic of PLCγ1:Gads:SLP76 interactions in the LAT condensate, where PLCγ1 activity is partially recovered compared to Grb2-only conditions.

Article Snippet: The membranes were incubated with either PLCγ1 (D9H10) XP rabbit mAb (Cell Signaling Technology, 1:1000 dilution), phospho-PLCγ1 (Tyr783) (D6M9S) rabbit mAb (Cell Signaling Technology, 1:1000 dilution), SLP-76 (D1R1A) rabbit mAb (Cell Signaling Technology, 1:1000 dilution), or Gads (UW40) mouse mAb conjugated with Alexa 647 (Santa Cruz Biotechnology, 1:200 dilution) overnight at 4 °C with agitation.

Techniques: Binding Assay, Membrane, Activity Assay