Journal: eLife

Article Title: Single cell analysis reveals multiple requirements for zinc in the mammalian cell cycle

doi: 10.7554/eLife.51107

Figure Lengend Snippet: Representative cell images for Hoescht DNA staining and p21 IF for WT MCF10A cells and p21 -/- MCF10A cells. p21 was detected with an anti-p21 antibody. Scale bars indicate 20 µm.

Article Snippet: Primary antibodies used were: p21 Waf1/Cip1 (CST, 2947S, Lot 10), pRB Ser807/811 (CST, D20B12 XP, Lot8), RPA2 (Abcam, ab2175, Lot GR3224197-5), 53BP1 (BD Biosciences, 612523, Lot 6217571) and secondaries were: AlexaFluor 488 (Abcam, ab150073, Lot GR328726-1), AlexaFluor 568 (Life Sciences A10042, Lot 1134929), or AlexaFluor 647 (Thermo Fisher Scientific, A21244, Lot 1156625).

Techniques: Staining

Journal: eLife

Article Title: Single cell analysis reveals multiple requirements for zinc in the mammalian cell cycle

doi: 10.7554/eLife.51107

Figure Lengend Snippet: ( A ) Single cell CDK2 traces of WT (top) and p21 -/- (bottom) MCF10A cells grown in either ZD, MM, or ZR media. Plots show n = 120 random traces selected from eight individual wells. CDK2 traces were aligned computationally to mitosis and colored by cell fate classifications. % total traces classified into each category are listed below traces (calculated from total traces n = 410, 3275, 3445 for WT cells grown in ZD, MM, and ZR, respectively and n = 441, 764, and 659 for p21 -/- cells). ( B ) CDK2 ratio vs. p21 intensity in WT MCF10A cells at a fixed timepoint after 40 hr of growth in ZD or MM media. p21 was detected with an anti-p21 antibody (for ZD, n = 412 cells pooled from three wells; for MM, n = 1414 cells pooled from three wells).

Article Snippet: Primary antibodies used were: p21 Waf1/Cip1 (CST, 2947S, Lot 10), pRB Ser807/811 (CST, D20B12 XP, Lot8), RPA2 (Abcam, ab2175, Lot GR3224197-5), 53BP1 (BD Biosciences, 612523, Lot 6217571) and secondaries were: AlexaFluor 488 (Abcam, ab150073, Lot GR328726-1), AlexaFluor 568 (Life Sciences A10042, Lot 1134929), or AlexaFluor 647 (Thermo Fisher Scientific, A21244, Lot 1156625).

Techniques:

Journal: eLife

Article Title: Single cell analysis reveals multiple requirements for zinc in the mammalian cell cycle

doi: 10.7554/eLife.51107

Figure Lengend Snippet:

Article Snippet: Primary antibodies used were: p21 Waf1/Cip1 (CST, 2947S, Lot 10), pRB Ser807/811 (CST, D20B12 XP, Lot8), RPA2 (Abcam, ab2175, Lot GR3224197-5), 53BP1 (BD Biosciences, 612523, Lot 6217571) and secondaries were: AlexaFluor 488 (Abcam, ab150073, Lot GR328726-1), AlexaFluor 568 (Life Sciences A10042, Lot 1134929), or AlexaFluor 647 (Thermo Fisher Scientific, A21244, Lot 1156625).

Techniques: Recombinant, Plasmid Preparation, Stable Transfection, Sequencing, Cloning, Staining, Software, Generated

Journal: Pathology and Oncology Research

Article Title: Modulated Electro-Hyperthermia Supports the Effect of Gemcitabine Both in Sensitive and Resistant Pancreas Adenocarcinoma Cell Lines

doi: 10.3389/pore.2021.1610048

Figure Lengend Snippet: Western blot analysis of p21 waf1 protein in Capan1 and Panc1 cells. The level of p21 waf1 increased significantly after mEHT + 0.5 nM GEM treatment in Capan1 cells at 48 h after treatment compared to the control, as seen on the representative WES protein loading (A,B) . In Panc1 cells, the level of p21 waf1 increased significantly at 24 h after treatment in mEHT + 12 nM GEM group compared to the control, p = 0.023, seen on the WES protein loading gel, with normalization to β-actin (C,E) . At 48 h the Kruskal-Wallis test showed a significant difference between the groups ( p = 0.028) with elevated levels of p21 waf1 in mEHT and 100 nM GEM monotherapies and in the mEHT + GEM groups, shown on representative WES protein loading images (D,F) .

Article Snippet: The following primary antibodies were used: rabbit polyclonal BAX (1:30, #HPA027878, Sigma-Aldrich, St. Louis, MO, United States), mouse monoclonal p21 waf1 (1:35, clone:70/Cip1/WAF1, #610234, BD Bioscience NJ 07417, United States), rabbit monoclonal Hsp27 (1:70, clone: D6W5V, #96357, Cell Signaling, Danvers, MA, United States), rabbit polyclonal Hsp70 (1:70, #4872, Cell Signaling, Danvers, MA, United States), rabbit monoclonal E-Cadherin (1:70, clone:EP700Y, #RM-2100, Thermo-Fisher, Cheshire, United Kingdom) and mouse monoclonal N-Cadherin (1:70, clone: 32/N-Cadherin, BD Bioscience NJ 07417, United States).

Techniques: Western Blot

Journal: Nature Communications

Article Title: Intracellular CD24 disrupts the ARF–NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation

doi: 10.1038/ncomms6909

Figure Lengend Snippet: ( a ) Heat-map depiction of alterations in gene expression in DU145 cells following CD24 gene silencing. RNA isolated from five independent cultures of Scr, three cultures of Sh1 and three cultures of Sh2 were compared using Affymeatrix Human U133 Plus 2.0 microarrays. Only those genes that met the statistical threshold of P <0.001 and mean alterations that reached the cutoff of >150% or<66% of Scr levels are displayed. The genes are listed in and the pathway analysis summary of affected genes is provided in . ( b ) CD24 silencing increases p21/CDKN1A mRNA. Error bars, s.d. n =3. The data have been reproduced three times. ( c ) CD24 silencing increased levels of p53 and p21 proteins, but not p27 protein. Representative images of western blots are shown and the data have been reproduced three times. ( d ) CD24 silencing reduces cell cycle re-entry after starvation. Scr, Sh1 and Sh2 clones were starved for 48 h in serum-free medium and then shifted into serum containing medium. The cells were harvested and analysed for cell cycle progression based on DNA content. These data have been reproduced three times. ( e ) Ectopic expression of CD24 in LNCaP cell line decreases levels of p53 and p21, but not p27. Representative images of western blots are shown and the data have been reproduced two times. ( f ) CD24 transduction increases proliferation of LNCaP cells. Data shown are triplicate means and s.d. of triplicates and have been reproduced twice. LSD, least significant difference.

Article Snippet: Antibodies specific for the following targets were used for this study: human CD24 (BD Pharmingen, ML5, Cat #:555426), mouse Cd24 (BD Pharmingen, M1/69, Cat #: 562360), CD24 (Santa Cruz Biotechnology, Cat #: sc-11406 and Cat #: sc-53660, and SN3b), NPM1 (Abcam, Cat #: ab10530), p14 (Santa Cruz Biotechnology, Cat #: sc-8340; Cell Signaling, Cat #:2407), p53 (Santa Cruz Biotechnology, DO-1, Cat #: sc-126; FL-393, Cat #sc-6243), MDM2 (Santa Cruz Biotechnology, Cat #: sc-7918), p21 (Cell Signaling, Cat #: 2946), p27 (Cell Signaling, Cat #: 2946), Cyclin B1 (Santa Cruz Biotechnology, Cat #: H-433), GFP (Cell Signaling, Cat #: 2946), Lamin B1 (Santa Cruz Biotechnology, sc-56145), H1.5 (Abcam ab18208), β-actin (Sigma-Aldrich, Cat #: A5441) and anti-IgG (Santa Cruz Biotechnology), phospho-ATM (Ser1981) (Rabbit mAb D6H9, Cat #5883, Cell Signaling) and ATM (Rabbit mAb D2E2, Cell Signaling, Cat #2873).

Techniques: Gene Expression, Isolation, Western Blot, Clone Assay, Expressing, Transduction

Journal: Nature Communications

Article Title: Intracellular CD24 disrupts the ARF–NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation

doi: 10.1038/ncomms6909

Figure Lengend Snippet: ( a ) Effect of CD24 silencing on expression of NPM, ARF, MDM2 and p53. Scrambled (Scr) or CD24 -shRNA-silenced DU145 cells were analysed for CD24, NPM, ARF, MDM2 and p53 by western blot. ( b ) In ARF-deficient U2OS cells, ectopic expression of CD24 had no effect on the levels of MDM2 and p53. Actin levels were used as loading control. ( c ) Endogenous mutant p53 is responsible for the upregulation of p21 in CD24-silenced DU145 cells. ( d ) In DU145 cells, silencing CD24 restores transcriptional induction of the p21 promoter. DU145 cells transduced with Scr or shRNA for either TP53 or CD24 were transfected with a luciferase reporter containing the p21 promoter. The WT reporter has an intact p53-binding site, whereas the mutant promoter has the site deleted. ( e , f ) CD24 silencing restores transcriptional activity of multiple TP53 mutants. DU145 cells in which the endogenous TP53 was silenced by shRNA were transduced with Scr or CD24 shRNA lentivirus. The stable cell lines were then transfected with p53 mutants in conjunction with WT or mutant p21 promoter reporter constructs. p53 levels ( e ) were determined by western blot, while luciferase activity ( f ) was measured at 72 h after transfection. The mutants used were p53 R273H , p53 V143A , p53 R280T and p53 R175H . Numbers on the x axis indicate the amino-acid positions of the p53 mutations. ( g ) As in e and f , except that the endogenous p21 and MDM2 mRNA were measured by quantitative PCR. Data shown are means and s.d. of triplicate samples. All data have been reproduced two or three times.

Article Snippet: Antibodies specific for the following targets were used for this study: human CD24 (BD Pharmingen, ML5, Cat #:555426), mouse Cd24 (BD Pharmingen, M1/69, Cat #: 562360), CD24 (Santa Cruz Biotechnology, Cat #: sc-11406 and Cat #: sc-53660, and SN3b), NPM1 (Abcam, Cat #: ab10530), p14 (Santa Cruz Biotechnology, Cat #: sc-8340; Cell Signaling, Cat #:2407), p53 (Santa Cruz Biotechnology, DO-1, Cat #: sc-126; FL-393, Cat #sc-6243), MDM2 (Santa Cruz Biotechnology, Cat #: sc-7918), p21 (Cell Signaling, Cat #: 2946), p27 (Cell Signaling, Cat #: 2946), Cyclin B1 (Santa Cruz Biotechnology, Cat #: H-433), GFP (Cell Signaling, Cat #: 2946), Lamin B1 (Santa Cruz Biotechnology, sc-56145), H1.5 (Abcam ab18208), β-actin (Sigma-Aldrich, Cat #: A5441) and anti-IgG (Santa Cruz Biotechnology), phospho-ATM (Ser1981) (Rabbit mAb D6H9, Cat #5883, Cell Signaling) and ATM (Rabbit mAb D2E2, Cell Signaling, Cat #2873).

Techniques: Expressing, shRNA, Western Blot, Control, Mutagenesis, Transduction, Transfection, Luciferase, Binding Assay, Activity Assay, Stable Transfection, Construct, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Intracellular CD24 disrupts the ARF–NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation

doi: 10.1038/ncomms6909

Figure Lengend Snippet: ( a ) By increasing the overall levels of p53 protein, Cd24 silencing increases the proportion of SV40 T antigen-unbound p53. SV40 T antigen was tagged with Flag and introduced into the mouse prostate cancer cell line C3 with or without transduction of CD24 shRNA. The total cellular (left panel) or SV40 T antigen-associated or non-associated p53 (right panel) were determined by either western blot or anti-Flag immunoprecipitation followed by western blot. Note that CD24 was not co-precipitated. S: supernatant; P: pellet. ( b ) Cd24 silencing reveals p53 function in SV40 large T antigen (SV40 Tag)-transduced (top) and human papilloma viral protein E6 (HPV E6)-transduced C3 cells (bottom). SV40 Tag- or HPV E6-transduced C3 cells were transfected with WT or p53-binding site mutant p21 promoter linked with a luciferase reporter. The function of the endogenous Cd24 and Tp53 locus was confirmed by shRNA silencing of either genes. Data shown are means and s.d. of relative luciferase activity. ( c ) In the Tp53 +/+ TRAMP model, targeted mutation of Cd24 increased the levels of p53 and p21. Data shown are photographs of immunohistochemical analysis of p21 and p53 levels from either Cd24 +/+ or Cd24 −/− prostate cancer samples. ( d ) Cd24 silencing increases the proportion of HPV E6-unbound p53, as in a , except that HPV E6 was used instead of SV40 TAg. All data in this figure have been reproduced two or three times. Co-IP, co-immunoprecipitation; Mut, mutant; WB, western blot.

Article Snippet: Antibodies specific for the following targets were used for this study: human CD24 (BD Pharmingen, ML5, Cat #:555426), mouse Cd24 (BD Pharmingen, M1/69, Cat #: 562360), CD24 (Santa Cruz Biotechnology, Cat #: sc-11406 and Cat #: sc-53660, and SN3b), NPM1 (Abcam, Cat #: ab10530), p14 (Santa Cruz Biotechnology, Cat #: sc-8340; Cell Signaling, Cat #:2407), p53 (Santa Cruz Biotechnology, DO-1, Cat #: sc-126; FL-393, Cat #sc-6243), MDM2 (Santa Cruz Biotechnology, Cat #: sc-7918), p21 (Cell Signaling, Cat #: 2946), p27 (Cell Signaling, Cat #: 2946), Cyclin B1 (Santa Cruz Biotechnology, Cat #: H-433), GFP (Cell Signaling, Cat #: 2946), Lamin B1 (Santa Cruz Biotechnology, sc-56145), H1.5 (Abcam ab18208), β-actin (Sigma-Aldrich, Cat #: A5441) and anti-IgG (Santa Cruz Biotechnology), phospho-ATM (Ser1981) (Rabbit mAb D6H9, Cat #5883, Cell Signaling) and ATM (Rabbit mAb D2E2, Cell Signaling, Cat #2873).

Techniques: Transduction, shRNA, Western Blot, Immunoprecipitation, Transfection, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Immunohistochemical staining, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Senescence-inhibitory Δ133p53α counteracts accelerated ageing and mortality

doi: 10.64898/2025.12.31.697195

Figure Lengend Snippet: Western blot analyses of lamin A/C and progerin, full-length p53, Δ133p53α, and p21 Waf1/Cip1 were performed in four 15-week-old Δ133p53α-expressing Group-1 mice ( CAG-133 Tam/+ ; Cre Tg/+ ; Lmna G609G/+ ), along with four each of age-matched, non-expressing control Group-2 ( CAG-133 LSL/+ ; Cre Tg/+ ; Lmna G609G/+ ) and Group-4 mice ( CAG-133 +/+ ; Cre +/+ ; Lmna G609G/+ ), as well as two age-matched wild-type mice ( CAG-133 +/+ ; Cre +/+ ; Lmna +/+ ). Results from skin ( a ), skeletal muscle ( b ), kidney ( c ), spleen ( d ), and lung ( e ) are presented. An inter-blot control (liver from a Group-1 mouse) was included in all blots. F, female; M, male. GAPDH was a loading control and used for normalization of p21 Waf1/Cip1 , progerin, and full-length p53 expression levels. Quantitative data summaries of p21 Waf1/Cip1 , progerin, and full-length p53 in Group-1, -2 and -4 mice are shown as relative values to Group-2 mice (mean ± s.d. from n = 4; open circles indicate two females, and closed circles indicate two males). P values were determined by Welch’s t -test. Two wild-type mice were used only as references and not for statistical comparisons.

Article Snippet: Primary antibodies used were as follows: anti-p53 antibodies SAPU (sheep polyclonal) and DO-11 (mouse monoclonal; Bio-Rad, MCA1704) ; anti-p21 Waf1/Cip1 (mouse monoclonal; Santa Cruz Biotechnology, sc-6246, clone F-5); anti-lamin A/C (mouse monoclonal; Santa Cruz Biotechnology, sc-376248); anti-GAPDH (mouse monoclonal; Santa Cruz Biotechnology, sc-166574); and anti-β-actin (mouse monoclonal; Thermo Fisher Scientific, MA1-91399).

Techniques: Western Blot, Expressing, Control

Journal: bioRxiv

Article Title: Senescence-inhibitory Δ133p53α counteracts accelerated ageing and mortality

doi: 10.64898/2025.12.31.697195

Figure Lengend Snippet: The tissue section slides of the aorta ( a-c ) and skin ( d-f ) were prepared from the same set of Group-1, Group-2 and Group-4 mice as in , as well as four age-matched wild-type mice (the same two as in additional two), followed by immunohistochemical (IHC) staining of p21 Waf1/Cip1 ( a,d ), p16 Ink4a ( b,e ), and γ-H2AX ( c,f ). Representative images are shown on the left (scale bars, 20 µm in a-c and 200 µm in d-f ). Insets in d-f show enlarged images of the boxed areas containing hair follicles (scale bars, 50 µm). TA, tunica adventitia; TM, tunica media; TI, tunica intima; Lu, Lumen; Epi, epidermis; D, dermis; dWAT, dermal white adipose tissue. Positively stained nuclei were counted in the tunica media containing vascular smooth muscle cells (VSMCs) ( a-c ) and in the dermis containing fibroblasts and hair follicles ( d-f ). Quantitative summaries are presented on the right (mean ± s.d. from n = 4; open circles, females; closed circles, males). P values were determined by Welch’s t -test. g, Serum IL-6 concentrations (pg ml -1 ) quantitated by ELISA in the same set of mice as above (mean ± s.d. from n = 4; open circles, females; closed circles, males). P values were calculated by Welch’s t -test. h, Il6 mRNA levels quantitated by qRT-PCR in the aorta, skin, heart, kidney, and spleen from the same set of mice as above (mean ± s.d. from n = 4; each with technical triplicate). P values were calculated by Welch’s t -test. Muscle and lung tissues were also tested, but the Ct values were not sufficiently high for reliable analysis.

Article Snippet: Primary antibodies used were as follows: anti-p53 antibodies SAPU (sheep polyclonal) and DO-11 (mouse monoclonal; Bio-Rad, MCA1704) ; anti-p21 Waf1/Cip1 (mouse monoclonal; Santa Cruz Biotechnology, sc-6246, clone F-5); anti-lamin A/C (mouse monoclonal; Santa Cruz Biotechnology, sc-376248); anti-GAPDH (mouse monoclonal; Santa Cruz Biotechnology, sc-166574); and anti-β-actin (mouse monoclonal; Thermo Fisher Scientific, MA1-91399).

Techniques: Immunohistochemical staining, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Senescence-inhibitory Δ133p53α counteracts accelerated ageing and mortality

doi: 10.64898/2025.12.31.697195

Figure Lengend Snippet: a,b , Histopathological examination of naturally aged Group-1 ( CAG-133 Tam/+ ; Cre Tg/+ ), Group-2 ( CAG-133 LSL/+ ; Cre Tg/+ ), and Group-4 ( CAG-133 +/+ ; Cre +/+ , wild-type) mice at 26-28 months of age (n = 4 each; open circles, females; closed circles, males). a, In H&E-stained sections of the aorta, numbers of VSMCs in the tunica media (per mm 2 ) were counted as in . Thickness of the tunica media (µm) was measured as in Extended Data Fig. 6c. Scale bars, 50 µm (top) or 200 µm (bottom). TA, tunica adventitia; TM, tunica media; TI, tunica intima; Lu, Lumen. b, In H&E-stained sections of the skin, thickness of the dermal white adipose tissue as in Extended Data Fig. 6f, as well as thickness of the dermis as in , was measured. Scale bars, 100 µm. Epi, epidermis; D, dermis; dWAT, dermal white adipose tissue. Data are presented as mean ± s.d. (n = 4) ( a,b ). P values were calculated using Welch’s t -test ( a,b ). c, Serum IL-6 concentrations (pg ml -1 ) in the same set of mice as in a and b were measured as in . Data are mean ± s.d. from n = 4 (Group 1, 2, and 4) or n = 8 (Groups 2 and 4 combined). P values were calculated by Welch’s t -test. d,e, Analysis of human Δ133p53α ( d ) and CDKN1A (p21 WAF1/CIP1 ) ( e ) expression levels in suprapubic skin tissue across age groups ranging from 20-29 to 70-79 years. Sample numbers (n) are shown in parentheses. Y-axis represents log 2 (TPM + 1). P values were calculated using Kruskal-Wallis test. In d , an outlier exceeding 10 in the 60-69 group is not shown but was included in the statistical analysis. f, Correlation analysis between Δ133p53α and CDKN1A expression. Spearman’s rho and P values are indicated (Spearman’s rank correlation test).

Article Snippet: Primary antibodies used were as follows: anti-p53 antibodies SAPU (sheep polyclonal) and DO-11 (mouse monoclonal; Bio-Rad, MCA1704) ; anti-p21 Waf1/Cip1 (mouse monoclonal; Santa Cruz Biotechnology, sc-6246, clone F-5); anti-lamin A/C (mouse monoclonal; Santa Cruz Biotechnology, sc-376248); anti-GAPDH (mouse monoclonal; Santa Cruz Biotechnology, sc-166574); and anti-β-actin (mouse monoclonal; Thermo Fisher Scientific, MA1-91399).

Techniques: Staining, Expressing

Journal: Scientific Reports

Article Title: Identification of a p53 target, CD137L , that mediates growth suppression and immune response of osteosarcoma cells

doi: 10.1038/s41598-017-11208-x

Figure Lengend Snippet: Pathway analysis for the p53-induced and p53-repressed genes.

Article Snippet: Anti-p53 monoclonal antibody (clone DO-7) and anti-p21 monoclonal antibody (clone EA10) were purchased from Merck Millipore.

Techniques: Significance Assay

Journal: Scientific Reports

Article Title: Identification of a p53 target, CD137L , that mediates growth suppression and immune response of osteosarcoma cells

doi: 10.1038/s41598-017-11208-x

Figure Lengend Snippet: Identification of CD137L as a bone-specific p53 target gene. ( a ) Induction of Cd137l among 24 tissues. Samples were categorized into 4 groups: (K) p53 −/− mice without irradiation, (W) p53 +/+ mice without irradiation, (KX) p53 −/− mice with irradiation and (WX) p53 +/+ mice with irradiation (n = 3 per group). We calculated the median FPKM value of WX/maximum FPKM value of the median FPKM value in K, KX or W for 24 tissues. ( b ) At 24 h after transfection with each siRNA, U2OS cells were treated with ADR (2 μg/ml for 2 h). At 36 h after treatment, whole cell extracts were subjected to western blotting with an anti-CD137L, anti-p21, anti-p53, or anti-β-actin antibody. siRNA against EGFP was used as a control. β-actin was used for normalization of the expression levels. These images were cropped from full-length blots (Supplementary Fig. ). ( c ) Immunohistochemical staining of Cd137l in mouse p53 α or p53 −/− calvaria with or without radiation exposure. Representative images from three tissues in each group are shown. Scale Bars (left) = 50 μm, Scale Bars (right) = 20 μm. Black arrowhead shows osteoblast.

Article Snippet: Anti-p53 monoclonal antibody (clone DO-7) and anti-p21 monoclonal antibody (clone EA10) were purchased from Merck Millipore.

Techniques: Irradiation, Transfection, Western Blot, Control, Expressing, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: MICAL2 acts through Arp3B isoform-specific Arp2/3 complexes to destabilize branched actin networks

doi: 10.1101/2020.09.21.306522

Figure Lengend Snippet: A In vitro pyrene actin assays using 12.5 nM of the indicated recombinant Arp2/3 reveal Arp3 and Arp3B containing complexes are equally efficient at nucleating actin regardless of the ARPC1A/ARPC5 and ARPC1B/ARPC5L background. B In vitro TIRF microscopy images to visualize branched actin formation. The top row shows image stills taken from TIRF assays using 2.5 nM Arp2/3 complexes containing Arp3B, ARPC1A and ARPC5 at the indicated times (see Movie S1) Scale bar = 15 μm. The bottom row shows the same panels after automatic detection of filament branches (yellow nodes) and ends (red nodes) with AnaMorf ImageJ plugin. C Quantification of mean branch number and length. D Quantification of the halflife of photoactivated GFP PA -β-actin in actin tails in Arp3B RNAi treated cells. These data were collected at same time as the data in . E Quantification of the half-life of Arp3 and Arp3B in vaccinia actin tails after activation of the GFP PA tag. The data in D and E from 3 independent experiments were combined and error bars represent SEM. for the indicated number of tails. Tukey’s multiple comparisons test was used to determine statistical significances, where **** P <0.0001.

Article Snippet: The other antibodies used in this study are listed below: ARPC2/p34-Arc polyclonal (Millipore, 07-227, 1:1000 dilution), ARPC5 monoclonal (Synaptic Systems, 305011, clone 323H3, 1:1000 dilution), Arp2 monoclonal (Abcam, ab129018, clone EPR7979, 1:1000 dilution), ARPC5L monoclonal (Abcam, ab169763, clone EPR10274, 1:1000 dilution), ARPC1A polyclonal (Sigma, HPA004334, 1:250 dilution), Arp3 monoclonal (Sigma, A5979, clone FMS338, 1:1000 dilution), ARPC1B polyclonal (Bethyl Laboratories, A302-781, 1:1000 dilution), ARPC3 monoclonal (BD Transduction labs, 612234, clone 26/p21-Arc, 1:1000 dilution), ARPC4 polyclonal (Sigma, SAB1100901, 1:1000 dilution), β-actin monoclonal (Sigma, A5316, clone AC-74, 1:1000 dilution), Vinculin monoclonal (Sigma, V4505, 1:1000 dilution), coronin-1C polyclonal (Invitrogen, PA5-21775, 1:500 dilution), a-Actinin monoclonal (Sigma, A5044, 1:1000 dilution), GAPDH monoclonal (Santa Cruz, sc-32233, 1:1000 dilution), Cortactin monoclonal (Millipore, 05-180, clone 4F11, 1:1000 dilution), MICAL1 polyclonal (Proteintech, 14818-1-AP, 1:1000 dilution), GFP monoclonal (custom made by CRUK, 1:1000 dilution).

Techniques: In Vitro, Recombinant, Microscopy, Activation Assay