Journal: Viruses

Article Title: Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry

doi: 10.3390/v17040542

Figure Lengend Snippet: siRNA-mediated knockdown of WAVE1 and WAVE2 inhibits HPV16 infection in HeLa cells. On day 0, HeLa cells were seeded and transfected with siRNA in a 6-well microplate. On day 2, cells were collected and seeded onto a 24-well microplate to establish technical replicates. On day 3, cells were infected with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid for 48 h. Protein expression for ( A ) WASP, ( C ) WAVE1, ( E ) WAVE2 or ( G ) WAVE1 and WAVE2 was measured via Western blotting on day 5. NC was the negative control siRNA used in this study, while S1, S2, and S3 refer to each of three separate siRNAs used to target the indicated proteins. For panels ( G , H ), S2 targeting WAVE1 and S3 targeting WAVE2 were employed to achieve knockdown of both proteins. Half volumes of each siRNA were combined for transfection so that the final concentration of siRNA in each experiment remained consistent. The percentage of HPV16 infected cells was also determined on day 5 (48 h post-infection) via flow cytometry for the knockdowns of ( B ) WASP, ( D ) WAVE1, ( F ) WAVE2, or ( H ) both WAVE1 and WAVE2. Each bar represents three biological replicates comprising technical triplicates and shows the mean %GFP+ cells ± standard deviation ( n = 3, normalized to WT). One-way ANOVA with Dunnett’s multiple comparison test was used to statistically determine significance (ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Anti-WAVE1 (PA5-78273), anti-WAVE2 (PA5-60975), anti-ITGβ4 (MA5-17104), anti-SERCA2 (MA3-919), anti-VPS25 (PA5-99005) anti-GAPDH (1D4), Texas Red-X goat anti-rabbit (T6391), Texas Red-X goat anti-mouse (T862), Alexa Fluor 488 goat anti-mouse (A11029), Alexa Fluor 488 goat anti-rabbit (A11034), and Alexa Fluor 680 goat anti-mouse (A21058) antibodies were purchased from ThermoFisher Scientific.

Techniques: Knockdown, Infection, Transfection, Plasmid Preparation, Expressing, Western Blot, Negative Control, Concentration Assay, Flow Cytometry, Standard Deviation, Comparison

Journal: Viruses

Article Title: Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry

doi: 10.3390/v17040542

Figure Lengend Snippet: WAVE1 knockout (W1KO), W2KO, and W1/W2KO alters cellular morphology, but not proliferation, and inhibits HPV16 infection in multiple cell lines. ( A ) WAVE1 (W1) WAVE2 (W2) or both (W1-W2KO) proteins were knocked out in wild-type (WT) HeLa cells via CRISPR/Cas9 and confirmed by Western blotting. ( B ) Representative phase-contrast images of WT, W1KO, W2KO, and W1–W2KO HeLa cells were taken on the FloID Cell Imaging Station (20× magnification, scale bar = 50 µm). Black arrows indicate narrow protrusions, while white arrows indicate lamellipodial protrusions. ( C ) W1KO, W2KO, and W1/W2KO HeLa cells were seeded in equal amounts, grown for 48 h, and then analyzed for differences in DNA quantity via CyQUANT cell proliferation assay (Thermo Fisher) compared to WT. WT, W1KO, W2KO, and W1/W2KO ( D ) HeLa or ( E ) B16-F1 cells were treated with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid. The percentage of infected cells (based on GFP reporter gene expression) was measured at 48 h post-infection via flow cytometry. Background from mock-infected cells was subtracted. For HeLa cells, at least 2 independent clones of each knockout were screened for consistent inhibition of HPV16 infection. Each bar represents three biological repeats comprising technical triplicates and shows DNA quantification over 48 h for panel ( C ) or the mean %GFP+ cells ± standard deviation ( n = 3, normalized to WT) for panels ( D , E ). One-way ANOVA with Dunnett’s multiple comparison test was used to statistically determine significance (ns = not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Anti-WAVE1 (PA5-78273), anti-WAVE2 (PA5-60975), anti-ITGβ4 (MA5-17104), anti-SERCA2 (MA3-919), anti-VPS25 (PA5-99005) anti-GAPDH (1D4), Texas Red-X goat anti-rabbit (T6391), Texas Red-X goat anti-mouse (T862), Alexa Fluor 488 goat anti-mouse (A11029), Alexa Fluor 488 goat anti-rabbit (A11034), and Alexa Fluor 680 goat anti-mouse (A21058) antibodies were purchased from ThermoFisher Scientific.

Techniques: Knock-Out, Infection, CRISPR, Western Blot, Imaging, CyQUANT Assay, Proliferation Assay, Plasmid Preparation, Gene Expression, Flow Cytometry, Clone Assay, Inhibition, Standard Deviation, Comparison

Journal: Viruses

Article Title: Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry

doi: 10.3390/v17040542

Figure Lengend Snippet: HPV infectivity is functionally recovered by WAVE1 or WAVE2 expression in HeLa cells. WT and ( A ) W1KO or ( C ) W2KO cells were transduced with a mammalian gene expression lentiviral control vector or a vector containing either GFP-WAVE1 or GFP-WAVE2, respectively (Vector Builder). Transduced cells received an antibiotic resistance gene and underwent selection. WT, ( B ) W1KO, ( D ) W2KO and cells with WAVE protein expression restored were treated with HPV16 PsVs (TCID 30 ) containing an RFP reporter plasmid. The percentage of infected cells (RFP reporter gene transduction) was measured at 48 h post-infection via flow cytometry. Background from mock infected cells was subtracted. Each bar represents three biological repeats comprising technical triplicates and show the mean %RFP+ cells ± standard deviation ( n = 3, normalized to WT). One-way ANOVA with Dunnett’s multiple comparison test was used to statistically determine significance (ns = not significant, **** p < 0.0001).

Article Snippet: Anti-WAVE1 (PA5-78273), anti-WAVE2 (PA5-60975), anti-ITGβ4 (MA5-17104), anti-SERCA2 (MA3-919), anti-VPS25 (PA5-99005) anti-GAPDH (1D4), Texas Red-X goat anti-rabbit (T6391), Texas Red-X goat anti-mouse (T862), Alexa Fluor 488 goat anti-mouse (A11029), Alexa Fluor 488 goat anti-rabbit (A11034), and Alexa Fluor 680 goat anti-mouse (A21058) antibodies were purchased from ThermoFisher Scientific.

Techniques: Infection, Expressing, Transduction, Gene Expression, Control, Plasmid Preparation, Selection, Flow Cytometry, Standard Deviation, Comparison

Journal: Viruses

Article Title: Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry

doi: 10.3390/v17040542

Figure Lengend Snippet: HPV16 colocalizes with actin and WAVE proteins at the cellular dorsal surface. WT HeLa cells expressing LifeAct-GFP seeded in chambered microscope slides were first cooled from 37 °C to 4 °C for 0.5 h to inhibit endocytosis prior to the addition of HPV16 L1L2 VLPs (10 ng/1 × 10 6 cells) in ice-cold media for 1 h. Cells were then returned to 37 °C for 10 min prior to fixation with 4% paraformaldehyde for 10 min at room temperature, which was the temperature for subsequent steps. Samples were then permeabilized with 0.1% Triton X-100, blocked with 1% BSA, and immunostained against HPV16 L1 and ( A ) WAVE1 or ( C ) WAVE2. Hoechst 33342 was added during secondary antibody addition as a counterstain. Z-stacked images were generated via laser scanning confocal microscopy. ( A , C ) depict maximum-intensity projections of Z-stacks of images depicting representative cells. The color channels are labeled at the upper left of each image. ( B ) and ( D ) depict the analysis of the spatial relationship between signals. To generate these images, we utilized Imaris 10.1.1 Microscopy Image Analysis Software (Oxford Instruments). Briefly, a “surface” was created for each signal, which is an Imaris segmentation algorithm. Surfaces were generated to provide object–object statistics. Parameters included the smoothing of surface details to 0.2 um with the method of absolute intensity thresholding. Background signal was subtracted through voxel size filtration (voxels smaller than 10 were excluded). Next, colocalization between channels was determined by the colocalization tool. Colocalized voxels (as determined by a Manders coefficient of 1) between surfaces were determined by first thresholding images to include true signals and restrict noise. New channels were then created of colocalization voxels. For both conditions, 3 fields containing 5–15 cells across 3 biological replicates were imaged. Scale = 10 µm.

Article Snippet: Anti-WAVE1 (PA5-78273), anti-WAVE2 (PA5-60975), anti-ITGβ4 (MA5-17104), anti-SERCA2 (MA3-919), anti-VPS25 (PA5-99005) anti-GAPDH (1D4), Texas Red-X goat anti-rabbit (T6391), Texas Red-X goat anti-mouse (T862), Alexa Fluor 488 goat anti-mouse (A11029), Alexa Fluor 488 goat anti-rabbit (A11034), and Alexa Fluor 680 goat anti-mouse (A21058) antibodies were purchased from ThermoFisher Scientific.

Techniques: Expressing, Microscopy, Generated, Confocal Microscopy, Labeling, Software, Filtration

Journal: Viruses

Article Title: Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry

doi: 10.3390/v17040542

Figure Lengend Snippet: Knockout of WAVE1, WAVE2, or both, prevents HPV16 stimulated HeLa cells from expressing dorsal surface actin protrusions. Cells were prepared as described in ; however, cells were not permeabilized during immunostaining. Untreated (top row, − symbol) or HPV16-infected WT, W1KO, or W1/W2KO HeLa cells (10 ng/1 × 10 6 cells) (middle row, + symbol) treated with CellLight Actin-GFP were imaged via laser scanning confocal microscopy to obtain Z-stacks. Z-stacks were then stitched together and rotated to view the XZ-oriented volume. Scale: images 1, 2, 4–7 = 10 µm; image 3 = 8 µm; image 8 = 14 µm. 22 cells were analyzed per condition.

Article Snippet: Anti-WAVE1 (PA5-78273), anti-WAVE2 (PA5-60975), anti-ITGβ4 (MA5-17104), anti-SERCA2 (MA3-919), anti-VPS25 (PA5-99005) anti-GAPDH (1D4), Texas Red-X goat anti-rabbit (T6391), Texas Red-X goat anti-mouse (T862), Alexa Fluor 488 goat anti-mouse (A11029), Alexa Fluor 488 goat anti-rabbit (A11034), and Alexa Fluor 680 goat anti-mouse (A21058) antibodies were purchased from ThermoFisher Scientific.

Techniques: Knock-Out, Expressing, Immunostaining, Infection, Confocal Microscopy

Journal: Viruses

Article Title: Human Papillomavirus Type 16 Stimulates WAVE1- and WAVE2-Dependent Actin Protrusions for Endocytic Entry

doi: 10.3390/v17040542

Figure Lengend Snippet: Knockout of WAVE1, WAVE2, or both results in a significant reduction of dorsal surface actin protrusions. Dorsal surface actin protrusions were quantified using the same method as described in . The graph depicts the average number of protrusions per cell ± standard deviation. Statistics: 2-way ANOVA with comparison of means was used to statistically determine significance, corrected for multiple comparisons using Tukey’s test (ns = not significant, **** p < 0.0001).

Article Snippet: Anti-WAVE1 (PA5-78273), anti-WAVE2 (PA5-60975), anti-ITGβ4 (MA5-17104), anti-SERCA2 (MA3-919), anti-VPS25 (PA5-99005) anti-GAPDH (1D4), Texas Red-X goat anti-rabbit (T6391), Texas Red-X goat anti-mouse (T862), Alexa Fluor 488 goat anti-mouse (A11029), Alexa Fluor 488 goat anti-rabbit (A11034), and Alexa Fluor 680 goat anti-mouse (A21058) antibodies were purchased from ThermoFisher Scientific.

Techniques: Knock-Out, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: WAVE1 and WAVE2 facilitate human papillomavirus-driven actin polymerization during cellular entry

doi: 10.1101/2024.10.28.620484

Figure Lengend Snippet: On day 0 HeLa cells were seeded and transfected with siRNA in a 6-well microplate. On day 2, cells were collected and seeded onto a 24-well microplate to establish technical replicates. On day 3, cells were infected with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid for 48 hours. Protein expression of relevant proteins was measured via Western blotting on day 5 (A, C, E, G). NC is the negative control siRNA used in this study, while S1, S2, and S3 refer to each of three separate siRNAs used to target the indicated proteins. For panels G and H, S2 targeting WAVE1 and S3 targeting WAVE2 were employed to achieve knockdown of both proteins. Half volumes of each siRNA were combined for transfection so that the final concentration of siRNA in each experiment remained consistent. The percentage of HPV16 infected cells was also determined on day 5 (48 hours post infection) via flow cytometry (B, D, F, H). Each bar represents three biological replicates comprised of technical triplicates and show the mean %GFP+ cells ± standard deviation (n=3, normalized to WT). 1-way ANOVA with Dunnett’s multiple comparisons test was used to statistically determine significance (ns=not significant, **p<0.001, ***p<0.0001, ****p<0.0001).

Article Snippet: Anti-WAVE1 (PA5- 78273), anti-WAVE2 (PA5-60975), anti-ITGβ4 (MA5-17104), anti-GAPDH (1D4), Texas Red-X goat anti-rabbit (T6391), Texas Red-X goat anti-mouse (T862), Alexa Fluor 488 goat anti-mouse (A11029), Alexa Fluor 488 goat anti-rabbit (A11034), and Alexa Fluor 680 goat anti-mouse (A21058) antibodies were purchased from ThermoFisher Scientific.

Techniques: Transfection, Infection, Plasmid Preparation, Expressing, Western Blot, Negative Control, Knockdown, Concentration Assay, Flow Cytometry, Standard Deviation

Journal: bioRxiv

Article Title: WAVE1 and WAVE2 facilitate human papillomavirus-driven actin polymerization during cellular entry

doi: 10.1101/2024.10.28.620484

Figure Lengend Snippet: (A) WAVE1 (W1) WAVE2 (W2) or both (DKO) proteins were knocked out in wild type (WT) HeLa cells via CRISPR/Cas9 and confirmed by Western blotting. (B) Representative phase-contrast images of WT, W1KO, W2KO, and W1/W2KO HeLa cells were taken on the FloID Cell Imaging Station (20x magnification, scale bar = 50μm). (C) W1KO, W2KO, and W1/W2KO HeLa cells were seeded in equal amounts, grown for 48 hours, and then analyzed for differences in DNA quantity via CyQUANT Cell Proliferation Assay (Thermo Fisher) compared to WT. (D and E) WT, W1KO, W2KO, and W1/W2KO HeLa or B16- F1 cells were treated with HPV16 PsVs (TCID 30 ) containing a GFP reporter plasmid. The percentage of infected cells (based on GFP reporter gene expression) was measured at 48 hours post infection via flow cytometry. Background from mock infected cells was subtracted. For HeLa cells, at least 2 independent clones of each knockout were screened for consistent inhibition of HPV16 infection. Each bar represents three biological repeats comprised of technical triplicates and show DNA quantification over 48 hours (Panel C) or the mean %GFP+ cells ± standard deviation (n=3, normalized to WT) (Panels D and E). 1-way ANOVA with Dunnett’s multiple comparisons test was used to statistically determine significance (ns=not significant, **p<0.001, ***p<0.0001, ****p<0.0001).

Article Snippet: Anti-WAVE1 (PA5- 78273), anti-WAVE2 (PA5-60975), anti-ITGβ4 (MA5-17104), anti-GAPDH (1D4), Texas Red-X goat anti-rabbit (T6391), Texas Red-X goat anti-mouse (T862), Alexa Fluor 488 goat anti-mouse (A11029), Alexa Fluor 488 goat anti-rabbit (A11034), and Alexa Fluor 680 goat anti-mouse (A21058) antibodies were purchased from ThermoFisher Scientific.

Techniques: CRISPR, Western Blot, Imaging, CyQUANT Assay, Proliferation Assay, Plasmid Preparation, Infection, Expressing, Flow Cytometry, Clone Assay, Knock-Out, Inhibition, Standard Deviation

Journal: bioRxiv

Article Title: WAVE1 and WAVE2 facilitate human papillomavirus-driven actin polymerization during cellular entry

doi: 10.1101/2024.10.28.620484

Figure Lengend Snippet: (A and C) WT and W1KO or W2KO cells were transduced with a mammalian gene expression lentiviral control vector or a vector containing either GFP-WAVE1 or GFP-WAVE2, respectively (Vector Builder). Transduced cells received an antibiotic resistance gene and underwent selection. (B and D) WT, KO, and cells with WAVE protein expression restored were treated with HPV16 PsVs (TCID 30 ) containing an RFP reporter plasmid. The percentage of infected cells (RFP reporter gene transduction) was measured at 48 hours post infection via flow cytometry. Background from mock infected cells was subtracted. Each bar represents three biological repeats comprised of technical triplicates and show the mean %RFP+ cells ± standard deviation (n=3, normalized to WT). 1-way ANOVA with Dunnett’s multiple comparisons test was used to statistically determine significance (ns=not significant, **p<0.001, ***p<0.0001, ****p<0.0001).

Article Snippet: Anti-WAVE1 (PA5- 78273), anti-WAVE2 (PA5-60975), anti-ITGβ4 (MA5-17104), anti-GAPDH (1D4), Texas Red-X goat anti-rabbit (T6391), Texas Red-X goat anti-mouse (T862), Alexa Fluor 488 goat anti-mouse (A11029), Alexa Fluor 488 goat anti-rabbit (A11034), and Alexa Fluor 680 goat anti-mouse (A21058) antibodies were purchased from ThermoFisher Scientific.

Techniques: Transduction, Expressing, Control, Plasmid Preparation, Selection, Infection, Flow Cytometry, Standard Deviation

Journal: bioRxiv

Article Title: WAVE1 and WAVE2 facilitate human papillomavirus-driven actin polymerization during cellular entry

doi: 10.1101/2024.10.28.620484

Figure Lengend Snippet: WT HeLa cells expressing LifeAct-GFP seeded in chambered microscope slides were first cooled from 37°C to 4°C for 0.5 h to inhibit endocytosis prior to the addition of HPV16 VLPs (10 ng/1E6 cells) in ice cold media for 1 hour. Cells were then returned to 37°C for 10 minutes prior to fixation with 4% paraformaldehyde for 10 minutes at room temperature, which was the temperature for subsequent steps. Samples were then permeabilized with 0.1% Triton X-100, blocked with 1% BSA, and immunostained against HPV16 L1 and (A) WAVE1 or (C) WAVE2. Hoescht 33342 was added during secondary antibody addition as a counterstain. Z-stacked images were generated via laser scanning confocal microscopy. (A and C) maximum intensity projections of Z-stacks of images depicting candidate cells. The color channels are labeled at the upper left of each image. (B and D) to analyze the spatial relationship between signals, we utilized Imaris 10.1.1 Microscopy Image Analysis Software (Oxford Instruments). Briefly, a “surface” was created for each signal, which is an Imaris segmentation algorithm. Surfaces were generated to provide object-object statistics. Parameters included the smoothing of surface details to 0.2 um with the method of absolute intensity thresholding. Background signal was subtracted through voxel size filtration (voxels smaller than 10 were excluded). Next, colocalization between channels was determined by the colocalization tool. Colocalized voxels (as determined by a Manders’ coefficient of 1) between surfaces were determined by first thresholding images to include true signals and restrict noise. New channels were then created of colocalization voxels. For both conditions, 3 fields containing 5-15 cells across 3 biological replicates were imaged. Scale = 10 µm.

Article Snippet: Anti-WAVE1 (PA5- 78273), anti-WAVE2 (PA5-60975), anti-ITGβ4 (MA5-17104), anti-GAPDH (1D4), Texas Red-X goat anti-rabbit (T6391), Texas Red-X goat anti-mouse (T862), Alexa Fluor 488 goat anti-mouse (A11029), Alexa Fluor 488 goat anti-rabbit (A11034), and Alexa Fluor 680 goat anti-mouse (A21058) antibodies were purchased from ThermoFisher Scientific.

Techniques: Expressing, Microscopy, Generated, Confocal Microscopy, Labeling, Software, Filtration

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 contributes to the ability of B cells to spread on immobilized anti-Ig. ( A ) A20 B-lymphoma cells and primary B cells were transfected with control siRNA or WAVE2 siRNA and analyzed via immunoblotting with Abs to WAVE2 or actin. WAVE2 band intensities were normalized to the actin loading control for the same sample and expressed relative to values for control siRNA-transfected cells. Full uncropped blots are shown in . ( B – D ) A20 cells were transfected with control siRNA or WAVE2 siRNA (WAVE2 KD) or pre-treated for 1 h with 100 µM CK-666. The cells were added to coverslips coated with 2.4 μg/cm 2 anti-IgG for the indicated times and then stained with rhodamine-phalloidin to visualize F-actin. Representative images are shown in panel ( B ). Scale bars: 10 µm. Cell areas were quantified using ImageJ version 10.9.0. Panel ( C ) shows one of three independent experiments with similar results. Each dot is one cell, and the medians and interquartile ranges are shown for >25 cells per condition. p -values were calculated using the Mann–Whitney U test. Panel D shows combined data from 3 independent experiments. Each symbol is an individual experiment and the data are presented as the mean ± SEM for the median values from the 3 experiments. p -values were calculated using two-tailed paired t -tests. ( E – G ) Control siRNA- and WAVE2 siRNA-transfected A20 cells were added to coverslips that had been coated with 2.4 μg/cm 2 anti-IgG for the indicated times and then stained with rhodamine-phalloidin. The data are presented as in panels ( B – D ). ( H – J ) Primary B cells transfected with control siRNA or WAVE2 siRNA (WAVE2 KD) were added to coverslips coated with 2.4 μg/cm 2 anti-IgM for the indicated times and then stained with rhodamine-phalloidin. The data are presented as in panels ( B – D ). All scale bars are 10 µm.

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques: Transfection, Control, Western Blot, Staining, MANN-WHITNEY, Two Tailed Test

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 KD does not reduce the expression of Hem1 protein and does not affect BCR signaling in response to immobilized anti-Ig. ( A ) A20 cells were transfected with control siRNA or WAVE2 siRNA, cultured for 48 h, and then analyzed via immunoblotting with Abs to WAVE2, Hem1, or actin (loading control). Normalized levels of WAVE2 or Hem1 relative to control siRNA-transfected cells are shown. ( B ) A20 cells that had been transfected with control siRNA or WAVE2 siRNA were allowed to spread on anti-IgG-coated tissue culture wells for the indicated times before analyzing BCR signaling by immunoblotting for the phosphorylation of the CD79a ITAM (pCD79a) or the phosphorylated (activated) forms of ERK (pERK) or Akt (pAkt). The same cell lysates were probed for total CD79a, ERK, or Akt. The dashed red line was overlaid on the images of the blots to visually separate the time courses for the control siRNA and WAVE2 siRNA-transfected cells. For both panels, one of three independent experiments with similar results is shown. Full uncropped blots are shown in .

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques: Expressing, Transfection, Control, Cell Culture, Western Blot, Phospho-proteomics

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 localizes to the peripheral actin ring in spreading B cells. ( A , B ) A20 cells were allowed to spread for 30 min on coverslips coated with 2.4 μg/cm 2 anti-Ig G. The cells were stained for F-actin and WAVE2 and then imaged via STED microscopy ( A ). Scale bar: 5 µm. Panel ( B ) shows F-actin and WAVE2 fluorescence intensity profiles along the yellow lines in panel ( A ). ( C , D ) Primary murine B cells that had been cultured overnight with IL-4 or with IL-4 + LPS were allowed to spread for 30 min on coverslips coated with 2.4 μg/cm 2 anti-IgM. The cells were stained for F-actin and WAVE2 and imaged via confocal microscopy. Enlarged images of the cells indicated by the yellow boxes on the merge panels are shown to the right. Scale bars: 10 µm. Panel ( D ) shows F-actin and WAVE2 fluorescence intensity profiles along the yellow lines in the enlarged cell images in panel ( C ). In the plot profiles in panels ( B , D ), the grey lines represent F-actin fluorescence intensity and the purple lines represent WAVE2 fluorescence intensity.

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques: Staining, Microscopy, Fluorescence, Cell Culture, Confocal Microscopy

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 contributes to peripheral F-actin assembly and actin retrograde flow. ( A ) Control siRNA- and WAVE2 siRNA-transfected A20 cells were allowed to spread on anti-IgG-coated coverslips for 15 or 30 min before being stained for F-actin and imaged via STED microscopy. Scale bar: 5 µm. ( B , C ) Confocal microscopy images of the control siRNA- and WAVE2 siRNA-transfected A20 cells from the experiments in B–D were used to calculate the average peripheral actin ring thickness for each cell, as described in the Methods section. Panel ( B ) shows representative data from a single experiment with the median and interquartile range for >20 cells per condition. p -values were calculated using the Mann–Whitney U test. Panel ( C ) shows the mean ± SEM for the median values from 3 independent experiments. p -values were calculated using two-tailed paired t -tests. ( D , E ) A20 cells that had been co-transfected with F-tractin-GFP and either control siRNA or WAVE2 siRNA were allowed to spread for 10 min on coverslips coated with 2.4 μg/cm 2 anti-IgG before initiating live-cell confocal microscopy imaging. Images were acquired at 1 s intervals for 2 min. In panel ( D ), representative kymographs along the yellow lines in the top panels (Scale bar: 10 µm) are shown in the bottom panels. The centripetal velocity (Δx/Δt) was calculated for individual actin tracks on the kymographs. Panel ( E ) shows two independent experiments in which the actin retrograde flow velocity was calculated for >10 tracks per cell for 3–6 cells. Each dot is one track. Medians and interquartile ranges are shown. The Mann–Whitney U test was used to calculate p -values.

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques: Control, Transfection, Staining, Microscopy, Confocal Microscopy, MANN-WHITNEY, Two Tailed Test, Imaging

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 KD does not reduce recruitment of the Arp2/3 complex to actin structures. Control siRNA- and WAVE2 siRNA-transfected A20 cells were allowed to spread on anti-IgG-coated coverslips for 15 or 30 min. The cells were stained for F-actin and the p34/ARPC2 subunit of the Arp2/3 complex and imaged via confocal microscopy ( A ) Representative images. Scale bars: 10 µm. ( B ) The confocal images were used to determine the Manders’ coefficient for the fraction of p34/ARPC2 that co-localized with F-actin. Super-plot showing combined data from 2 independent experiments for >25 cells per condition. Each dot is one cell and the different colors represent the two independent experiments. The large symbols represent the median values for each experiment. p -values for control cells versus WAVE2 KD cells in the combined experiments were calculated using the Mann–Whitney U test.

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques: Control, Transfection, Staining, Confocal Microscopy, MANN-WHITNEY

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 contributes to the LFA-1-dependent formation of actomyosin arcs. ( A ) A20 cells were allowed to spread for 10 or 30 min on coverslips coated with a suboptimal density of anti-IgG (0.625 µg/cm 2 ), ICAM-1 (0.15 µg/cm 2 ), or 0.625 µg/cm 2 anti-IgG plus 0.15 µg/cm 2 ICAM-1. The cells were stained for F-actin and cell areas were quantified from confocal microscopy images. Medians and interquartile ranges are shown for >25 cells per condition. p -values were calculated using the Mann–Whitney U test. ( B ) Control and WAVE2 KD A20 cells were allowed to spread for 30 min on coverslips coated with a low density of anti-IgG (0.625 µg/cm 2 ) with or without 0.15 µg/cm 2 ICAM-1. The means ± SEM for the median values from 3 independent experiments are shown. p -values were calculated using two-tailed paired t -tests. ( C ) A20 cells that had been transfected with a plasmid encoding myosin IIA-GFP were allowed to spread for 30 min on coverslips that had been coated with 2.4 µg/cm 2 anti-IgG (high anti-IgG) or 0.625 µg/cm 2 anti-IgG (low anti-IgG) plus 0.15 µg/cm 2 ICAM-1. Representative images are shown. Scale bar: 10 µm. ( D , E ) Control siRNA- and WAVE2 siRNA-transfected A20 cells were allowed to spread for 30 min on coverslips that had been coated with 2.4 µg/cm 2 anti-IgG (high anti-IgG) or 0.625 µg/cm 2 anti-IgG (low anti-IgG) plus 0.15 µg/cm 2 ICAM-1. Cells were stained for F-actin and imaged by STED microscopy. Representative images are shown, and the yellow arrowheads indicate actin arcs ( D ). For comparison, examples are shown of WAVE2 KD cells that did or did not form actin arcs when plated on low anti-IgG plus ICAM-1. Scale bar: 5 µm. In panel ( E ), the percent of cells that formed distinct actin arcs was determined in 3 independent experiments (n > 30 cells per condition). The data are presented as the mean ± SEM for the 3 experiments. p -values were calculated using two-tailed paired t -tests.

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques: Staining, Confocal Microscopy, MANN-WHITNEY, Control, Two Tailed Test, Transfection, Plasmid Preparation, Microscopy, Comparison

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 contributes to the ability of B cells to spread on FN. ( A , B ) A20 cells that had been transfected with either control siRNA or WAVE2 siRNA, or pre-treated for 1 h with 100 µM CK-666, were added to FN-coated coverslips. After the indicated times the cells were fixed, stained with rhodamine-phalloidin, and imaged via confocal microscopy (( A ) scale bar: 10 µm) or STED microscopy (( B ) scale bar: 5 µm). Representative images are shown. ( C , D ) In each experiment, cell areas were quantified from confocal microscopy images for >30 cells per condition. A single representative experiment ( C ) as well as combined data from 3 independent experiments ( D ) are shown. The data are presented in as . In panel ( D ), p -values were calculated for the control cells versus WAVE2 KD cells using two-tailed paired t -tests. Where no error bars were visible, they were smaller than the symbols.

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques: Transfection, Control, Staining, Confocal Microscopy, Microscopy, Two Tailed Test

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 enhances APC-induced BCR signaling and signal amplification. Control siRNA- and WAVE2 siRNA-transfected A20 cells were added to COS-7 cells expressing the ani-Igκ surrogate Ag on their surface. After 5–30 min, the cells were fixed, permeabilized, and stained for pCD79 and the surrogate Ag. ( A ) Representative confocal images of clustered Ag and proximal BCR signaling (pCD79) at the B cell-COS-7 cell contact site. Scale bar: 10 µm. ( B – E ) For each B cell, the total amount of clustered pCD79 (panels B , C ) and clustered Ag were quantified for >50 cells per condition and were used to determine the ratio of clustered pCD79/clustered Ag (signal amplification; panels D , E ). Panels ( B , D ) show the data from the same representative experiment. Each dot is one cell, and the medians and interquartile ranges are shown. p -values were determined using the Mann–Whitney U test. Panels ( C , E ) show combined data from 3 independent experiments. Each red symbol is an individual experiment, and the data are presented as the mean ± SEM for the median values from the 3 experiments. p -values were calculated using two-tailed paired t -tests. In panels ( C , E ) the dashed lines represent the values for control cells, which were defined as 100%, and the areas shaded in blue highlight time points for which the values for WAVE2 KD cells were significantly different ( p < 0.005) from those for the control cells.

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques: Amplification, Control, Transfection, Expressing, Staining, MANN-WHITNEY, Two Tailed Test

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 is important for APC-induced B cell activation. Control siRNA- and WAVE2 siRNA-transfected primary B cells, as well as primary B cells that had been treated with CK-666 for 1 h, were added to COS-7 cells expressing the anti-Igκ surrogate Ag. The cells were co-cultured overnight before being stained for CD69 and IgM and analyzed via flow cytometry. ( A ) After gating on IgM + cells, forward/side scatter (top row) was used to identify single live B cells and quantify their CD69 fluorescence (bottom row). ( B ) The surrogate APC-induced increase in cell surface CD69 levels (left panel; geometric means) and percent CD69 + cells were calculated by subtracting the values for unstimulated B cells that were cultured without anti-Igκ-expressing COS-7 cells. Each colored dot is an independent experiment. The data are presented as the mean + SEM for the 3 experiments. p -values were calculated using the One-Way repeated measures ANOVA test.

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques: Activation Assay, Control, Transfection, Expressing, Cell Culture, Staining, Flow Cytometry, Fluorescence

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 regulates B cell responses. By activating the Arp2/3 complex, the WAVE2-containing WRC contributes to the BCR- and integrin-induced actin remodeling that promotes cell spreading, actomyosin arc formation, and APC-induced BCR signaling. Created with BioRender . The red arrows represent downstream signaling from the BCR and integrins. For the images at the bottom of the figure, the integrin-induced spreading image is taken from B, the image showing actomyosin arcs is taken from D (the yellow arrowheads point to the actomyosin arcs), and the image for APC-induced BCR signaling is taken from A.

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques:

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 KD does not reduce the expression of Hem1 protein and does not affect BCR signaling in response to immobilized anti-Ig. ( A ) A20 cells were transfected with control siRNA or WAVE2 siRNA, cultured for 48 h, and then analyzed via immunoblotting with Abs to WAVE2, Hem1, or actin (loading control). Normalized levels of WAVE2 or Hem1 relative to control siRNA-transfected cells are shown. ( B ) A20 cells that had been transfected with control siRNA or WAVE2 siRNA were allowed to spread on anti-IgG-coated tissue culture wells for the indicated times before analyzing BCR signaling by immunoblotting for the phosphorylation of the CD79a ITAM (pCD79a) or the phosphorylated (activated) forms of ERK (pERK) or Akt (pAkt). The same cell lysates were probed for total CD79a, ERK, or Akt. The dashed red line was overlaid on the images of the blots to visually separate the time courses for the control siRNA and WAVE2 siRNA-transfected cells. For both panels, one of three independent experiments with similar results is shown. Full uncropped blots are shown in .

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques: Expressing, Transfection, Control, Cell Culture, Western Blot, Phospho-proteomics

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 KD does not reduce the expression of Hem1 protein and does not affect BCR signaling in response to immobilized anti-Ig. ( A ) A20 cells were transfected with control siRNA or WAVE2 siRNA, cultured for 48 h, and then analyzed via immunoblotting with Abs to WAVE2, Hem1, or actin (loading control). Normalized levels of WAVE2 or Hem1 relative to control siRNA-transfected cells are shown. ( B ) A20 cells that had been transfected with control siRNA or WAVE2 siRNA were allowed to spread on anti-IgG-coated tissue culture wells for the indicated times before analyzing BCR signaling by immunoblotting for the phosphorylation of the CD79a ITAM (pCD79a) or the phosphorylated (activated) forms of ERK (pERK) or Akt (pAkt). The same cell lysates were probed for total CD79a, ERK, or Akt. The dashed red line was overlaid on the images of the blots to visually separate the time courses for the control siRNA and WAVE2 siRNA-transfected cells. For both panels, one of three independent experiments with similar results is shown. Full uncropped blots are shown in .

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques: Expressing, Transfection, Control, Cell Culture, Western Blot, Phospho-proteomics