rabbit anti arp3  (Proteintech)

Journal: bioRxiv

Article Title: Simulated Microgravity Enhances Adipocyte Maturation and Glucose Uptake via Increased Cortical Actin Remodeling

doi: 10.1101/2024.01.30.578049

Figure Lengend Snippet: (a) Ras homolog gene family member A ( RHOA) and Rho-associated coiled-coil kinases ( ROCK1) gene expression was significantly increased in sµg (n=5 biological replicates). (b) Total RhoA protein expression was not affected in 1g and sµg conditions (n=3 biological replicates, the cropped blot is shown, the full-length blot is presented in Supplementary Fig. S1). (c) pCofilin/Cofilin protein expression was increased in sµg conditions (n=6 biological replicates, the cropped blot is shown, the full-length blot is presented in Supplementary Fig. S2). (d) Actin related protein 3 (ARP3) protein expression was significantly increased in sµg (n=3 biological replicates, the cropped blot is shown, the full-length blot is presented in Supplementary Fig. S1). (e) ARP2 protein expression was not affected in 1g and sµg conditions (n=3 biological replicates, the cropped blot is shown, the full-length blot is presented in Supplementary Fig. S2). (f) Simulated microgravity enhances adipocyte maturation via increasing ARP3 protein expression and enhancing cortical actin remodeling. Data are presented as means ± SEM. Comparisons between groups and statistical analysis were performed using an unpaired t-test with two-tailed p-values (*p < 0.05).

Article Snippet: For protein detection, the following primary antibodies (Cell Signaling Technology) were used: rabbit anti-actin related protein 3 (ARP3) (1:1000, Cat# 4738, RRID:AB_2221973), rabbit anti-ARP2 (1.500, Cat# 3128, RRID:AB_2181763), rabbit anti-ras homolog family member A (RhoA) (1:1000, Cat# 2117, RRID:AB_10693922), mouse anti-Akt (1:1000, Cell Signaling, Cat# 2920, RRID:AB_1147620), phosphorylated Akt (pAkt-Ser473) (1:1000 Cat# 4060, RRID:AB_2315049), rabbit anti-cofilin (1:500, Cat# 3318, RRID:AB_2080595), rabbit anti-phosphorylated cofilin (1:500, pcofilin-Ser3, Cat# 3311, RRID:AB_330238), and rabbit anti-beta actin (1:1000, Cat# 4970, RRID:AB_2223172), as a housekeeping protein.

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Simulated Microgravity Enhances Adipocyte Maturation and Glucose Uptake via Increased Cortical Actin Remodeling

doi: 10.1101/2024.01.30.578049

Figure Lengend Snippet: (a) ARP3 expression was significantly increased in mature adipocytes (n=6 biological replicates). (b) pAkt/Akt protein expression was not affected in both conditions after insulin stimulation (n=3 biological replicates, data are normalized to 1g (-ins) condition, cropped blot is shown, the full-length blot is presented in Supplementary Fig. S3). (c) Insulin-stimulated glucose uptake was significantly downregulated in the presence of CK-666 in sµg conditions (n=3 biological replicates). (d) Representative cross-sectional images of adipocytes in the absence (control, top panel) or the presence of CK-666 (bottom panel) in sµg, white arrow points to the brighter cytoplasmic fluorescent signal for GLUT4 in the presence of CK-666 (scale bar-20 µm). (e) GLUT4 translocation to the cell membrane was significantly decreased in the presence of CK-666 (n=4 biological replicates, 10-12 cells were quantified for each replicate). (f) Representative cross-sectional images of adipocytes in the absence (top panel) or the presence of CK-666 (bottom panel) in sµg, white arrow points to the brighter cytoplasmic fluorescent signal for F-actin in the presence of CK-666 (scale bar-20 µm). (g) F-actin cortex to cytoplasm ratio was significantly decreased in the presence of CK-666 (n=3 biological replicates, 10-12 cells were quantified for each replicate). (h) ARP3 inhibition prevented cortical actin remodeling; therefore, GLUT4 translocation to the cell membrane and decreased insulin-stimulated glucose uptake. Data are presented as means ± SEM. Comparisons between groups and statistical analysis were performed using two-way ANOVA with Tukey post hoc test, Mann– Whitney test, or unpaired t-test with two-tailed p-values (*p<0.05, ***p < 0.001, ****p<0.0001).

Article Snippet: For protein detection, the following primary antibodies (Cell Signaling Technology) were used: rabbit anti-actin related protein 3 (ARP3) (1:1000, Cat# 4738, RRID:AB_2221973), rabbit anti-ARP2 (1.500, Cat# 3128, RRID:AB_2181763), rabbit anti-ras homolog family member A (RhoA) (1:1000, Cat# 2117, RRID:AB_10693922), mouse anti-Akt (1:1000, Cell Signaling, Cat# 2920, RRID:AB_1147620), phosphorylated Akt (pAkt-Ser473) (1:1000 Cat# 4060, RRID:AB_2315049), rabbit anti-cofilin (1:500, Cat# 3318, RRID:AB_2080595), rabbit anti-phosphorylated cofilin (1:500, pcofilin-Ser3, Cat# 3311, RRID:AB_330238), and rabbit anti-beta actin (1:1000, Cat# 4970, RRID:AB_2223172), as a housekeeping protein.

Techniques: Expressing, Translocation Assay, Membrane, Inhibition, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Simulated Microgravity Enhances Adipocyte Maturation and Glucose Uptake via Increased Cortical Actin Remodeling

doi: 10.1101/2024.01.30.578049

Figure Lengend Snippet: (a) Ras homolog gene family member A ( RHOA) and Rho-associated coiled-coil kinases ( ROCK1) gene expression was significantly increased in sµg (n=5 biological replicates). (b) Total RhoA protein expression was not affected in 1g and sµg conditions (n=3 biological replicates, the cropped blot is shown, the full-length blot is presented in Supplementary Fig. S1). (c) pCofilin/Cofilin protein expression was increased in sµg conditions (n=6 biological replicates, the cropped blot is shown, the full-length blot is presented in Supplementary Fig. S2). (d) Actin related protein 3 (ARP3) protein expression was significantly increased in sµg (n=3 biological replicates, the cropped blot is shown, the full-length blot is presented in Supplementary Fig. S1). (e) ARP2 protein expression was not affected in 1g and sµg conditions (n=3 biological replicates, the cropped blot is shown, the full-length blot is presented in Supplementary Fig. S2). (f) Simulated microgravity enhances adipocyte maturation via increasing ARP3 protein expression and enhancing cortical actin remodeling. Data are presented as means ± SEM. Comparisons between groups and statistical analysis were performed using an unpaired t-test with two-tailed p-values (*p < 0.05).

Article Snippet: To detect ARP3 and GLUT4, the following primary antibodies were used: rabbit anti ARP3 (1:100, Cell Signaling Technology, Cat# 4738, RRID: AB_2221973), and mouse anti-GLUT4 (1:100, Cell Signaling Technology, Cat# 2213, RRID: AB_823508).

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Simulated Microgravity Enhances Adipocyte Maturation and Glucose Uptake via Increased Cortical Actin Remodeling

doi: 10.1101/2024.01.30.578049

Figure Lengend Snippet: (a) ARP3 expression was significantly increased in mature adipocytes (n=6 biological replicates). (b) pAkt/Akt protein expression was not affected in both conditions after insulin stimulation (n=3 biological replicates, data are normalized to 1g (-ins) condition, cropped blot is shown, the full-length blot is presented in Supplementary Fig. S3). (c) Insulin-stimulated glucose uptake was significantly downregulated in the presence of CK-666 in sµg conditions (n=3 biological replicates). (d) Representative cross-sectional images of adipocytes in the absence (control, top panel) or the presence of CK-666 (bottom panel) in sµg, white arrow points to the brighter cytoplasmic fluorescent signal for GLUT4 in the presence of CK-666 (scale bar-20 µm). (e) GLUT4 translocation to the cell membrane was significantly decreased in the presence of CK-666 (n=4 biological replicates, 10-12 cells were quantified for each replicate). (f) Representative cross-sectional images of adipocytes in the absence (top panel) or the presence of CK-666 (bottom panel) in sµg, white arrow points to the brighter cytoplasmic fluorescent signal for F-actin in the presence of CK-666 (scale bar-20 µm). (g) F-actin cortex to cytoplasm ratio was significantly decreased in the presence of CK-666 (n=3 biological replicates, 10-12 cells were quantified for each replicate). (h) ARP3 inhibition prevented cortical actin remodeling; therefore, GLUT4 translocation to the cell membrane and decreased insulin-stimulated glucose uptake. Data are presented as means ± SEM. Comparisons between groups and statistical analysis were performed using two-way ANOVA with Tukey post hoc test, Mann– Whitney test, or unpaired t-test with two-tailed p-values (*p<0.05, ***p < 0.001, ****p<0.0001).

Article Snippet: To detect ARP3 and GLUT4, the following primary antibodies were used: rabbit anti ARP3 (1:100, Cell Signaling Technology, Cat# 4738, RRID: AB_2221973), and mouse anti-GLUT4 (1:100, Cell Signaling Technology, Cat# 2213, RRID: AB_823508).

Techniques: Expressing, Translocation Assay, Membrane, Inhibition, MANN-WHITNEY, Two Tailed Test

Journal: PLoS ONE

Article Title: Myosin VI Regulates Actin Structure Specialization through Conserved Cargo-Binding Domain Sites

doi: 10.1371/journal.pone.0022755

Figure Lengend Snippet: False-colored confocal images of Arp3 (A, green) or cortactin (B, green), actin (phalloidin, red) and merged images in a myosin VI mutant background are shown. GFP-myosin VI is not shown in this figure Bar, 10 µm.

Article Snippet: Affinity-purified rabbit anti-Arp3 antibody or mouse anti-cortactin antibody (4F11, Millipore, Billerica, MA) were used and visualized using anti-rabbit or mouse secondary antibody conjugated to Alexa-568, respectively.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: MLL1 regulates cytokine-driven cell migration and metastasis

doi: 10.1101/2022.10.18.512715

Figure Lengend Snippet: (a) Epigenetic factors in the TCGA basal breast cancer dataset were queried for correlation with genes reported to be essential for cell migration genes, including RHOA, RAC1, ROCK1, ROCK2, MYLK, CDC42, LIMK1, ACTR2,ACTR3, and WASF3. MLL1 was the seventh-highest correlated gene among the 715 epigenetic factors and had high correlation with genes involved in myosin-based contractility. (b) Cells were embedded in 3D collagen gels and cells were tracked overnight using live-cell phase contrast microscopy. Resulting videos were analyzed to obtain cell trajectories, velocity, and diffusivity using the APRW cell-migration model. (c) shMLL1 cells exhibited shorter trajectories than scrambled non-targeting shRNA control and wild type cells. (d) shMLL1 cells had lower velocity, compared to control and WT cells. Velocities are depicted in a box-and-whisker plot, with each dot corresponding to one cell. The upper limit of the box represents the 75 th percentile, lower limit represents the 25 th percentile, and the center line represents the median. The top and bottom whiskers stretch to the 99 th and 1 st percentile, respectively. n(cells) = 123(WT), 122(Scr. cont.), 128(shMLL1 KD1), 139(KD2), 132(KD3); one-way ANOVA (**** p < 0.0001, F (4, 639) = 26.65); two-tailed t-test (**** p(SC - sh) < 0.0001, NS p(WT - SC) = 0.8151). (e) Pharmacological inhibition of MLL1-Menin interaction using MI-2-2 decreased cell velocity to levels similar to shMLL1 cells. n(cells) = 112(WT), 175(10 µM), 183(30 µM); one-way ANOVA (**** p < 0.0001, F (2, 467) = 78.12); two-tailed t-test (**** p < 0.0001). (f) MLL1 depletion impairs cancer cell proliferation. n(wells) = 11(each condition); two-tailed t-test (**** p < 0.0001). (g) shMLL1 tumors grew slower compared to scrambled control tumors. n(mice) = 7(each condition); one-way ANOVA (**** p < 0.0001, F (3, 24) = 35.18). (h) NSG mice bearing shMLL1 tumors survive longer than mice bearing scrambled control tumors. n(mice) = 7(each condition); Log rank (Mantel–Cox) test (*** p = 0.0001, HR = 0.20, 95% C.I. = 0.01 – 0.17). (i) H&E staining of lungs show darker staining for scrambled control tumors indicative of higher metastatic burden compared to shMLL1 tumors. (j) Quantification of total metastatic burden per lung showed an increased metastatic burden in mice bearing scrambled control tumors compared to shMLL1 tumor bearing mice. n(lungs) = 5(each condition, 1 lung/mouse); two-tailed t-test (** p = 0.0091). (k) Histogram of lesion size of a single lung shows that decreased metastatic burden in shMLL1 lungs were due to both reduced number of lesions as well as their decreased size. n(lesions) = 367 (Scr. cont.), 61 (shMLL1); two-tailed t-test (**** p < 0.0001). (l) RNA-seq analysis of MLL1-Menin-inhibited cells revealed downregulation of key cell migration and metastasis-related pathways including the epithelial-mesenchymal transition, IL-6-JAK- STAT3 signaling, and TGF-β signaling pathways. Data in this figure was generated with MDA-MB-231 cells in vivo (g-k) or embedded in 3D collagen gels (b-f and l) except panel a (TCGA).

Article Snippet: Primary antibodies were purchased from Cell Signaling technology (β-Actin-HRP #5125, GAPDH #5174, MLL1 Carboxy-terminal Antigen #14197, Menin #6891, Phospho-Stat3 (Tyr705) #9145, ARP3 #4738, Phospho-Myosin Light Chain 2 (Ser19) #3671, Phospho-Myosin Light Chain 2 (Thr18/Ser19) #3674, Tri-Methyl-Histone H3 (Lys4) #9751, ROCK1 #4035, ROCK2 #8236, NF-κB p65 #8242, and NF-κB1 p105/p50 #3035), or Novus Bio (Gli-2, #NB600) and used at manufacturer’s recommended concentration for western blotting.

Techniques: Migration, Microscopy, shRNA, Whisker Assay, Two Tailed Test, Inhibition, Staining, RNA Sequencing Assay, Generated, In Vivo

Journal: bioRxiv

Article Title: MLL1 regulates cytokine-driven cell migration and metastasis

doi: 10.1101/2022.10.18.512715

Figure Lengend Snippet: (a) Conditioned medium was collected from scrambled control cells and added to shMLL1 or scrambled control cells. Conditioned medium from scrambled control cells fully rescued cell migration of shMLL1 cells as seen in (b) trajectories and (c) velocities. n(cells) = 81(Scr. cont.), 81(Scr. Cont.+CM), 89 (shMLL1), 85 (shMLL1+CM); one-way ANOVA (**** p < 0.0001, F (3, 332) = 13.68); two-tailed t-test (**** p(sh - sh+CM) < 0.0001, NS p(SC - sh+CM) = 0.8563, NS p(SC+CM - sh+CM) = 0.4392, NS p(SC - SC+CM) = 0.3979). (d) MLL1-Menin inhibition leads to downregulation of key genes in the IL- 6/JAK/STAT3 signaling pathway assessed via qRT-PCR. MLL1-Menin inhibition by n(wells) = 4(each condition); one-way ANOVA (IL6: **** p < 0.0001, F (2, 9) = 61.73; STAT3: * p = 0.0104, F (2, 9) = 7.905; NFKB1: ** p = 0.0075, F (2, 9) = 8.858; NFKB2: **** p < 0.0001, F (2, 9) = 47.67 ; ACTR2: ** p = 0.0073, F (2, 9) = 8.918). (e) MI-2-2 [n(wells) = 4(each condition); one-way ANOVA (**** p < 0.0001, F (2, 9) = 41.11); two-tailed t-test (** p(0 - 10) = 0.0049, **** p(0 - 30) < 0.0001, ** p(10 - 30) = 0.005)] or (f) MI-503 decreased IL-6 production TNBC cell lines. n(wells) = 4(each condition); two-tailed t-test (** p(MDA-MB-231) = 0.0020, * p(BT-549) = 0.0420, *** p(SUM-149) = 0.0007, ** p(SUM-159) = 0.0030,). (g) Phase contract images of cells treated with MLL1-Menin inhibitor show decreased generation of protrusions. Protrusion generation can be rescued by adding IL-6+IL-8 to cells treated with MLL1-Menin inhibitor, MI-2-2. Scale bar 20 µm. (h) A temporal heatmap of protrusion-related parameters show that the cumulative number of protrusions generated per cell and the maximum length of protrusion generated by a cell were reduced by MLL1-Menin inhibition, which coincided with a decrease in cell migration (mean squared displacement). Each row is one cell, and each block is one condition. (i) A rescue of cell motility was attempted by supplementing cells with IL-6/8 on top of MLL1 inhibition by 10 µM MI-2-2. IL-6/8 supplementation fully rescued cell motility despite continuing inhibition of the MLL1-Menin interaction, evident in (j) velocity and (k) trajectories. n(cells) = 135(Untreat.), 82(IL), 98(MI), 120 (IL+MI); one-way ANOVA (**** p < 0.0001, F (3, 431) = 9.233); two-tailed t-test (**** p(MI – MI+IL) < 0.0001, **** p(Untreat. – MI) < 0.0001, NS p(Untreat. – MI+IL) = 0.2905, NS p(MI+IL – IL) = 0.8257, NS p(Untreat. – IL) = 0.4676). (l) Western blotting on pSTAT3 (downstream of IL-6/8) and Arp2/3 (involved in actin assembly downstream of STAT3) shows inhibition and rescue following MLL1 inhibition and IL-6/8 supplementation, respectively. (m) Quantification of four biological repeats of IL- 6/8 rescue western blots emphasize that pSTAT3 and Arp3 levels are significantly reduced and subsequently restored. n(western blots) = 4 (biological replicates); paired two-tailed t-test (** p(pSTAT3) = 0.0056, ** p(Arp3) = 0.0098). (n) STAT3 knockdown reduces cell migration to levels observed with MLL1-Menin inhibition. n(cells) = 110(Scr. cont.), 121(shSTAT3), 121(MI-2-2); one-way ANOVA (**** p < 0.0001, F (2, 349) = 28.75); two-tailed t-test (**** p(SC – shSTAT3) < 0.0001, NS p(shSTAT3 – MI-2-2) = 0.6118, **** p(SC – MI-2-2) < 0.0001). (o) Motility rescued via IL-6/8 supplementation was lost by inhibiting STAT3 (using S3I-201), which lies downstream of IL-6/8. n(cells) = 146(Untreat.), 134(MI), 146 (S3I), 107(MI+IL), 147(MI+IL+S3I); one-way ANOVA (**** p < 0.0001, F (4, 675) = 30.68); two-tailed t-test (**** p(Untreat. - MI) < 0.0001, **** p(MI – MI+IL) < 0.0001, **** p(MI+IL – MI+IL+S3I) < 0.0001, NS p(Untreat. – MI+IL) = 0.9059, NS p(S3I - MI+IL+S3I) = 0.0535). (p) Schematic illustration of MLL1-Menin-based regulation of cell migration. MLL1-Menin interaction controls the production of IL-6, which binds to the IL-6 receptor and leads to phosphorylation of STAT3. pSTAT3 drives actin filament assembly via Arp2/3. Data in this figure was generated with MDA-MB-231 cells embedded in 3D collagen gels except panel f (MDA-MB-231, BT549, SUM-149, and SUM-159).

Article Snippet: Primary antibodies were purchased from Cell Signaling technology (β-Actin-HRP #5125, GAPDH #5174, MLL1 Carboxy-terminal Antigen #14197, Menin #6891, Phospho-Stat3 (Tyr705) #9145, ARP3 #4738, Phospho-Myosin Light Chain 2 (Ser19) #3671, Phospho-Myosin Light Chain 2 (Thr18/Ser19) #3674, Tri-Methyl-Histone H3 (Lys4) #9751, ROCK1 #4035, ROCK2 #8236, NF-κB p65 #8242, and NF-κB1 p105/p50 #3035), or Novus Bio (Gli-2, #NB600) and used at manufacturer’s recommended concentration for western blotting.

Techniques: Migration, Two Tailed Test, Inhibition, Quantitative RT-PCR, Generated, Blocking Assay, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: F-Actin Dysplasia Involved in Organ of Corti Deformity in Gjb2 Knockdown Mouse Model

doi: 10.3389/fnmol.2021.808553

Figure Lengend Snippet: Significant increase of Vinculin was observed in the P0 KD group at P6. Protein expression level of Vinculin, Arp2 (A) , Arp3 (D) , and RHOA (G) in P0 control and P0 KD groups. Comparison of Vinculin (B) , Arp2 (C) , Arp3 (E) , and RHOA (F) by western blot in P0 control and P0 KD groups. The differences of Arp2, Arp3, and RHOA expression level between the P0 KD and the P0 control group were not significant ( P > 0.05). *Significantly different from the control group ( P < 0.05).

Article Snippet: Proteins were then detected using the following antibodies: anti-GAPDH rabbit monoclonal antibody (ANT324; Antgene, Biotechnology Company Ltd, Wuhan, China); anti-β-actin rabbit polyclonal antibody (ANT322, Antgene); anti-gamma-actin rabbit polyclonal antibody (11227-1-AP; Proteintech, Rosemont, IL, USA); anti-Cx26 rabbit polyclonal antibody (71-0500; Invitrogen, Carlsbad, CA, USA); anti-H3 histone rabbit antibody (A2348; ABclonal, Woburn, MA, USA); anti-α-catenin rabbit antibody (A19004, ABclonal); anti-LRRK2 rabbit polyclonal antibody (A17253, ABclonal); anti-RHOA rabbit antibody (A17253, ABclonal); anti-Arp2 rabbit antibody (A5734, ABclonal); anti-Arp3 rabbit antibody (A1046, ABclonal); anti-Vinculin rabbit antibody (A14193, ABclonal); anti-β-catenin rabbit antibody (YT0676, Immunoway, Plano, TX, USA); anti-p120-ctn rabbit antibody (12180-1-AP, Proteintech); anti-α-catenin rabbit antibody (YT0676, Immunoway); the dilution ratio of all solutions containing primary antibodies was 1:1,000.

Techniques: Expressing, Control, Comparison, Western Blot