Journal: Molecular Oncology

Article Title: ABI1 ‐based expression signature predicts breast cancer metastasis and survival

doi: 10.1002/1878-0261.13175

Figure Lengend Snippet: ABI1 expression alteration is associated with copy number alteration (CNA) and high‐aggressive basal‐like breast cancer. Box Plots: (A) Putative ABI1 DNA copy number alteration (CNA) drives ABI1 transcription level in subpopulations of primary breast cancer patients . The gene expression, CNA, tumor samples, and clinical datasets representing 1904 primary breast cancer samples were downloaded from METABRIC dataset ( https://www.cbioportal.org/ ). CNA categorization is the following: shallow deletion: 1 ( n = 166), diploid: 2 ( n = 1554), gain: 3 (146), and amplification: 4 ( n = 38). One‐way ANOVA test (Statistica 13) showed significant differences in the ABI1 expression between the groups and also in the entire cohort ( P < 1.00E‐9). Furthermore, the transcription level of ABI1 is highly significant and positively correlated with CNA (( r = 0.338; P < 1.00E‐6; estimated by Spearman). (B) ABI1 transcription level positively correlated with histologic grades (univariate and bivariate linear regression models testing shown significance at P < 1.00E‐6), however (C) negatively correlated with ER status. Bivariate linear regression models (Statistica 13) showed that both expression ABI1 expression level and CNA are significant ( r = −0.278; P < 1.0.00E‐6 and r = −0.207, P < 1.00E‐6 respectively); however, the ABI1 expression provides a major contribution in the bivariate linear regression function). Correlate coefficients in (B) and (C) were calculated by Kendall. (D) ABI1 overexpression is associated with basal‐like and claudin‐low breast cancer subtypes and aggressiveness of breast cancer scoring also by histologic grade. PAM50 (Basal‐like, HER2(+), luminal B, luminal A, normal‐like), and claudin‐low subtypes were ranked‐order according to the trend of decreasing of ABI1 expression. One‐way ANOVA test (Statistica 13) showed significant differences in the ABI1 expression between basal‐like, claudin‐low subtypes and other subtypes ( P < 1.00E‐6). ABI1 expression in the HER2 subtype was significantly higher than in luminal B or luminal A tumor subtypes ( P < 1.00E‐6) and higher but less significant than in the normal‐like tumor subtype ( P = 001). A negative trend in the ABI1 expression across rank‐ordered tumor subtypes was mostly defined by relative overexpression of Basal‐like and claudin‐low tumor subtypes; it was highly significant (one‐way ANOVA; P < 1.00E‐9; Statistica 13).

Article Snippet: Blots were probed with the following primary antibodies: ABI1 (Rockland, Pottstown, PA, USA; 1 : 1000), ABI2 (P‐20, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 500), ABI3 (GeneTex, Irvine CA, USA; 1 : 1000), WAVE1 (K91/36, MilliporeSigma, Burlington, MA, USA; 1 : 1000), WAVE2 (H‐110, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 1000), WAVE3 (W4642, Sigma‐Aldrich, St. Louis, MO, USA; 1 : 1000), or β‐actin (AC‐15, Sigma‐Aldrich; 1 : 10 000).

Techniques: Expressing, Gene Expression, Amplification, Over Expression

Journal: Molecular Oncology

Article Title: ABI1 ‐based expression signature predicts breast cancer metastasis and survival

doi: 10.1002/1878-0261.13175

Figure Lengend Snippet: ABI1 ‐based prognostic signature predicts disease‐free and metastatic‐free survival risks. The disease‐free survival (DFS) and disease metastasis‐free survival (DMFS) of patients stratified based on the ABI1 ‐associated signature derived by our survival prognostic analysis method (see Methods for details) is shown using Kaplan–Meier survival curves for Rosetta (A, C) and MetaData cohorts (B, D). The Wald statistic P ‐value and hazard ratio (HR) associated with the partitioning of the patients into distinct risk groups are also shown (see methods for details). Our method computationally categorizes each covariate (expression level of a gene) as a binarizing risk factor and stratifies each patient according to the multivariate expression pattern of the genes included in the signature (Table ). In panels A, B, C, and D: black color line = ‘low‐risk’, red = ‘intermediate risk’, blue = ‘high‐risk’ groups. Panels E and F represent the overall survival (OS) time functions for the patients with metastasis detected after diagnostic and following surgical treatment. The black color line is associated with the group of patients with relatively better disease outcomes, while the red color is associated with patients with poor disease outcomes. The tables at the bottom of plots show the number of patients who survived in the predicted groups more than the given time point.

Article Snippet: Blots were probed with the following primary antibodies: ABI1 (Rockland, Pottstown, PA, USA; 1 : 1000), ABI2 (P‐20, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 500), ABI3 (GeneTex, Irvine CA, USA; 1 : 1000), WAVE1 (K91/36, MilliporeSigma, Burlington, MA, USA; 1 : 1000), WAVE2 (H‐110, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 1000), WAVE3 (W4642, Sigma‐Aldrich, St. Louis, MO, USA; 1 : 1000), or β‐actin (AC‐15, Sigma‐Aldrich; 1 : 10 000).

Techniques: Derivative Assay, Expressing, Diagnostic Assay

Journal: Molecular Oncology

Article Title: ABI1 ‐based expression signature predicts breast cancer metastasis and survival

doi: 10.1002/1878-0261.13175

Figure Lengend Snippet: Abi1 loss does not impact the long‐term development of healthy mouse mammary glands. (A) Whole‐mount analysis of the inguinal mammary gland stained with Carmine Alum reveals no gross changes in gland anatomy at 5, 7, or 12 weeks of age after CRE‐mediated deletion of Abi1 . Morphometry of whole mounts reveals a significant increase in the number of terminal end buds in homozygous ABI1 null glands (B); however, this does not affect the elongation of the ductal tree (C) or the number of ductal branches (D). Scale bar, 5.0 mm. (E) Histological staining of mammary gland sections reveals no changes in tissue organization after CRE‐mediated loss of Abi1 . Scale bar, 100 μm. (F) Immunostaining of mammary sections using markers for luminal epithelial cells (CK8) and myoepithelial cells (CK14) reveals sustained organization of the ductal epithelium in both control and ABI1 null mammary glands. Scale bar, 50 μm. Error bars indicate SEM. (* indicates P < 0.05, Student’s t ‐test; n = 5 animals/genotype). (G) WB analysis indicates enhanced expression of Abi1 in mammary epithelium of Abi1(fl;fl) PyMT mice vs. Abi1 floxed mice Abi1 (fl;fl). Each lane represents one mammary gland (Abi1 fl;fl) or tumor [PyMT: Abi1(fl/fl)], ( n = 3 mice).

Article Snippet: Blots were probed with the following primary antibodies: ABI1 (Rockland, Pottstown, PA, USA; 1 : 1000), ABI2 (P‐20, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 500), ABI3 (GeneTex, Irvine CA, USA; 1 : 1000), WAVE1 (K91/36, MilliporeSigma, Burlington, MA, USA; 1 : 1000), WAVE2 (H‐110, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 1000), WAVE3 (W4642, Sigma‐Aldrich, St. Louis, MO, USA; 1 : 1000), or β‐actin (AC‐15, Sigma‐Aldrich; 1 : 10 000).

Techniques: Staining, Immunostaining, Control, Expressing

Journal: Molecular Oncology

Article Title: ABI1 ‐based expression signature predicts breast cancer metastasis and survival

doi: 10.1002/1878-0261.13175

Figure Lengend Snippet: Gene expression variability upon Abi1 depletion in primary PyMT breast cancer tumors defined by RNA‐seq.

Article Snippet: Blots were probed with the following primary antibodies: ABI1 (Rockland, Pottstown, PA, USA; 1 : 1000), ABI2 (P‐20, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 500), ABI3 (GeneTex, Irvine CA, USA; 1 : 1000), WAVE1 (K91/36, MilliporeSigma, Burlington, MA, USA; 1 : 1000), WAVE2 (H‐110, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 1000), WAVE3 (W4642, Sigma‐Aldrich, St. Louis, MO, USA; 1 : 1000), or β‐actin (AC‐15, Sigma‐Aldrich; 1 : 10 000).

Techniques: Gene Expression

Journal: Molecular Oncology

Article Title: ABI1 ‐based expression signature predicts breast cancer metastasis and survival

doi: 10.1002/1878-0261.13175

Figure Lengend Snippet: Abi1 KO severely impacts WAVE complex gene expression dynamics. (A) Western blot analysis of primary mammary tumors from Abi1 KO PyMT mice shows significant depletion of ABI1 protein, but only in the homozygote Abi1 null is there significant upregulation of ABI2 protein as indicated by densitometry (B). Each lane represents one mammary tumor isolated from one animal of that genotype. Error bars indicate SEM. ( P < 0.05, t ‐test; n = 3 animals/genotype). (C) Analysis of primary tumor histology reveals no significant changes in tumor grade between controls and Abi1 knockouts, ( P > 0.05, t ‐test; n ≥ 5 mice per genotype of age between 20 and 22 weeks were used for analysis, Table ). Error bars indicate SEM). (D) Immunostaining with antibodies against WAVE complex proteins supports our findings that ABI2 is upregulated only in ABI1 null breast tumors. WAVE1 retained its low expression, while WAVE2 was concomitantly depleted with ABI1, in agreement with WB data, above. 20× magnification; inset, 40× magnification, Scale bar, 50 μm.

Article Snippet: Blots were probed with the following primary antibodies: ABI1 (Rockland, Pottstown, PA, USA; 1 : 1000), ABI2 (P‐20, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 500), ABI3 (GeneTex, Irvine CA, USA; 1 : 1000), WAVE1 (K91/36, MilliporeSigma, Burlington, MA, USA; 1 : 1000), WAVE2 (H‐110, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 1000), WAVE3 (W4642, Sigma‐Aldrich, St. Louis, MO, USA; 1 : 1000), or β‐actin (AC‐15, Sigma‐Aldrich; 1 : 10 000).

Techniques: Gene Expression, Western Blot, Isolation, Immunostaining, Expressing

Journal: Molecular Oncology

Article Title: ABI1 ‐based expression signature predicts breast cancer metastasis and survival

doi: 10.1002/1878-0261.13175

Figure Lengend Snippet: Primary tumor growth kinetics analysis indicates Abi1 gene dose effect in heterozygous mice. (A) Primary tumor latency in PyMT animals is not significantly affected upon Abi1 KO. The X ‐axis of a panel (a) represents latency time comparison of the tumors in four treatment conditions defined on the upright corner of the panel (Abi1 fl/fl Cre‐, n = 14 mice; Abi1 fl/fl Cre+, n = 20 mice, Abi1 fl/wt Cre‐, n = 16; Abi1 fl/wt Cre+, n = 16 mice). (B‐E) Treatment effects of Abi1 disruption (fw Cre(+) vs fw Cre(‐) and tumor kinetics of tumor size in heterozygous or homozygous miceGraphical tools of Statistica‐13 were used. Each plot on panels (B‐E) shows tumor size at seven‐time points (w0, w1, w3, w3, w4, w5, and w6 (see Methods)) (for Abi1 fl/fl Cre+, or Cre‐, n = 13 mice were used; for Abi1 fl/wt Cre+, or Cre‐, n = 11 mice were used). The line connects start (Cre(‐)) with the endpoint (Cre(+)) tumor size datasets allowing the comparison of tumor kinetic observations to be easily followed; mean values of tumor size are linked by direct lines at the same detection time point. Wilks lambda statistics and Fisher test were used for estimation of treatment significance. Panels (B) and (C) represent a visualization of the treatment effect (Cre(‐) v.s. Cre(+)) of Abi1 on tumor size in observed time points. Vertical bars indicate 0.95 intervals, CI. An effective decomposition method of Statistica‐13 was used. The primary tumor size comparison in fastly growing mouse groups shows the exponential growth kinetics. (Methods, Table ). To compare gene dosage effects within heterozygote and homozygote groups, mean values in 7 observed time points were compared (see Table for details). Our results showed that in the cases of fast kinetics datasets, differences between the paired sample mean values were not significant for homozygote ( t ‐test, P > 0.15) but significant for heterozygote state ( t ‐test, P = 0.017). (See Methods and Table ).

Article Snippet: Blots were probed with the following primary antibodies: ABI1 (Rockland, Pottstown, PA, USA; 1 : 1000), ABI2 (P‐20, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 500), ABI3 (GeneTex, Irvine CA, USA; 1 : 1000), WAVE1 (K91/36, MilliporeSigma, Burlington, MA, USA; 1 : 1000), WAVE2 (H‐110, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 1000), WAVE3 (W4642, Sigma‐Aldrich, St. Louis, MO, USA; 1 : 1000), or β‐actin (AC‐15, Sigma‐Aldrich; 1 : 10 000).

Techniques: Comparison, Disruption

Journal: Molecular Oncology

Article Title: ABI1 ‐based expression signature predicts breast cancer metastasis and survival

doi: 10.1002/1878-0261.13175

Figure Lengend Snippet: Abi1 gene knockout reduces metastatic burden in heterozygous and homozygous mice. Representative tumor kinetics of primary (panels a‐b) vs metastatic tumors (panels c‐d). Panel (A) Comparison of the primary tumor volume kinetics in Abi1 homozygous KO mouse (fl/fl; Cre+) (G209, data: red triangle; best‐fit function: red line) and the control Abi1 (fl/fl Cre‐) mouse (G184, data: blue circle; best‐fit function: blue line). (B) Comparison of the primary tumor kinetics of Abi1 KO heterozygous (fl/wt Cre+) mouse (G251, data: pink triangle; best‐fit function: pink line) and the Abi1 control (fl/wt; Cre‐) mouse (G202, data: green circle; best‐fit function: green line). Kinetics of mean values (A and B) were fitted by exponential curve f ( t ; a , b ) = ( a − b ) * exp( at ), where t is time, constant a is the rate of cell population growth and constant ( a − b ) is the initial tumor population size. Each kinetic dataset includes seven time points (see also Table ). The estimated parameters in Abi1 fl/fl Cre (‐) tumors: a = 0.77 +/− 0.159, t ‐test, P = 0.0047, b = 0.2 +/− 0.678, t ‐test, P > 0.1 and in Abi1 fl/fl Cre (+) a = 0.60 +/− 0.153, t ‐test, P = 0.0039), b = 0.1 +/− 0.586, t ‐test, P > 0.1. Estimated parameters in Abi1 fl/wt Cre (‐) tumors: a = 0.79 +/− 0.091, t ‐test, P = 0.001), b =−1.00 +/− 0.964, t ‐test, P > 0.1, and in Abi1 fl/wt Cre (+) a = 0.589 +/− 0.110, t ‐test, P = 0.0031, b =−0.30 +/− 0.66, t ‐test, P > 0.1. According to these results, differences between mean values of the tumor sizes in the studied groups in time are not significant. While primary tumor volume kinetics was not significantly different in these mice vs. their corresponding controls, (A, homozygous ABI1 KO vs. control) and (B, heterozygous KO vs. control), the difference in metastatic tumor burden of the same mice within each mouse genotype was significant (C) and (D). Panels (C) and (D) show the frequency distributions of a lung metastatic foci size in the heterozygous and homozygous mice, which primary tumors kinetics showed on panels (A) and (B), respectively. Each Y‐axis value shown in the histograms (C‐D) represents a count of metastatic foci within a metastatic size normalized interval (a bin). The bin was defined by rounding the metastatic size divided by 1000 to the nearest integer, and the number of metastatic foci in each bin was counted. Based on our findings, the metastases size frequency distribution in the lung has skewed form with the long right tail. To provide a visualization of such frequency distribution, we used log 10 − log 10 plot. We used the same color for dots of the empirical distributions and the fitting function lines, as was indicated in the figures. Such empirical frequency distribution was modeled and parameterized using the shifted log‐normal distribution function: f ( x ; y 0 , x 0 , a , b ) = y 0 + a ∗ exp ( ‐ 0.5 ∗ ( ln ( x / x 0 ) / b ) 2 ) , where x is the node size and y 0 , x 0 , a , b unknown parameters. We estimated the parameters using the nonlinear curve fitting option of SigmaPlot‐13 software. Datasets and detailed results of the parameterization of this function are presented in Table . (E‐F) show histogram bar plots for the distribution of the average number of metastases foci size in the lungs of Abi1 KO mice in comparison to their genetic controls. X‐axis indicates binning for every 5000 μm 2 metastasis colony area size, with bin 1 representing 0–5000 μm 2 and bin 21 representing 100001 μm 2 and larger; Y‐axis: count of the samples within given binning interval (+/− SEM). The size stratification of individual metastatic colonies shows that mice lacking ABI1 still have relatively small metastatic colonies but they grow slowly or/and stay at dormant state and appear unable to establish macrometastases when compared to our controls ( P < 0.001; Wilcoxon signed‐rank test). Lung metastasis quantification was performed following fixation, paraffin embedding and sectioning: three 5μm sections (sectioned every 50μm) were collected from each mouse (Abi1 fl/fl, Cre‐, n = 7; Abi1 fl/fl, Cre+, n = 6; Abi1 fl/wt, Cre‐, n = 6; Abi1 fl/wt, Cre+, n = 6; animals per genotype, age 18–22 weeks), were stained with hematoxylin and eosin, and imaged using Omnyx digital pathology scanner (GE Healthcare). Images were quantified using ImageJ software (NIH). Results of panels (E) and (F) support the results presented in (C) and (D). (G) Histological staining of representative lung sections reveals severely diminished metastasis upon deletion of the Abi1 gene. Scale bar, 1 mm. Inset, 4× magnification.

Article Snippet: Blots were probed with the following primary antibodies: ABI1 (Rockland, Pottstown, PA, USA; 1 : 1000), ABI2 (P‐20, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 500), ABI3 (GeneTex, Irvine CA, USA; 1 : 1000), WAVE1 (K91/36, MilliporeSigma, Burlington, MA, USA; 1 : 1000), WAVE2 (H‐110, Santa Cruz Biotechnology, Dallas, TX, USA; 1 : 1000), WAVE3 (W4642, Sigma‐Aldrich, St. Louis, MO, USA; 1 : 1000), or β‐actin (AC‐15, Sigma‐Aldrich; 1 : 10 000).

Techniques: Gene Knockout, Comparison, Control, Software, Staining

Journal: PLoS ONE

Article Title: The Spectrin Cytoskeleton Is Crucial for Adherent and Invasive Bacterial Pathogenesis

doi: 10.1371/journal.pone.0019940

Figure Lengend Snippet: ( a ) HeLa cells were infected with EPEC and immunolocalized with antibodies to spectrin, adducin and p4.1 together with probes to actin and DAPI. Arrowheads indicate areas of interest that are found in the insets. ( b) Immunolocalization of spectrin to sites of wild-type (WT) EPEC attachment and to EPEC effector mutants: Δ escN , Δ tir , Δ tir : tir , Δ tir : tir Y474F. ( c) Western blot of siRNA treated HeLa cells targeted against spectrin (Spectrin RNAi) and non-targeting control pool siRNA (CP). Calnexin was used as a loading control. Immunofluorescent image of spectrin RNAi, attached bacteria show they are unable to form pedestals. ( d ) Quantification of the number of bacteria forming pedestals. For each treatment, 3 independent experiments were run; for microscopy counts n = 3 for each experiment, error bars show s.e.m. Statistics were not run due to a complete absence of pedestals in infected RNAi samples Scale bars are 5 µm.

Article Snippet: Antibodies used in this study included a mouse monoclonal anti-β-Spectrin II antibody (used at 2.5 μg/ml for immunofluorescence and 0.25 μg/ml for western blots) (Becton Dickinson), rabbit anti-α-adducin (used at 2 μg/ml for immunofluorescence and 0.2 μg/ml for western blots) (Santa Cruz), rabbit anti-EPB41 (protein 4.1) (used at 1.7 μg/ml for immunofluorescence and 0.17 μg/ml for western blots) (Sigma), rabbit anti-calnexin (Becton Dickinson) (used at 1∶2000).

Techniques: Infection, Western Blot, Control, Bacteria, Microscopy

Journal: PLoS ONE

Article Title: The Spectrin Cytoskeleton Is Crucial for Adherent and Invasive Bacterial Pathogenesis

doi: 10.1371/journal.pone.0019940

Figure Lengend Snippet: HeLa cells were infected with EPEC and were immunolocalized with antibodies against spectrin, adducin and p4.1 as well as actin and DAPI. Arrows indicate areas of interest that are found in the insets. Scale bars are 5 µm.

Article Snippet: Antibodies used in this study included a mouse monoclonal anti-β-Spectrin II antibody (used at 2.5 μg/ml for immunofluorescence and 0.25 μg/ml for western blots) (Becton Dickinson), rabbit anti-α-adducin (used at 2 μg/ml for immunofluorescence and 0.2 μg/ml for western blots) (Santa Cruz), rabbit anti-EPB41 (protein 4.1) (used at 1.7 μg/ml for immunofluorescence and 0.17 μg/ml for western blots) (Sigma), rabbit anti-calnexin (Becton Dickinson) (used at 1∶2000).

Techniques: Infection

Journal: PLoS ONE

Article Title: The Spectrin Cytoskeleton Is Crucial for Adherent and Invasive Bacterial Pathogenesis

doi: 10.1371/journal.pone.0019940

Figure Lengend Snippet: ( a ) Immunolocalization of spectrin cytoskeletal elements, spectrin, adducin and p4.1 with actin and DAPI during S . Typhimurium SL1344 invasion in HeLa cells. Actin labeling identified invasion sites with actin-rich membrane ruffles. ( b ) Images illustrating the inability of a Δ sipA mutant to recruit spectrin to sites of invasion. Δ sipA : sipA restored spectrin recruitment to invasion sites. ( c ) Spectrin localization to sites of internalized S . Typhimurium SL1344-GFP. Areas of interest are indicated by arrowheads and highlighted in insets. ( d ) Quantifying invasion efficiency after depletion of spectrin with siRNA as compared to a non-targeting control pool (CP). For each treatment, 3 independent experiments were run; for microscopy counts n = 3, error bars show s.e.m, *P<0.0001 for all statistics. Scale bars are 5 µm.

Article Snippet: Antibodies used in this study included a mouse monoclonal anti-β-Spectrin II antibody (used at 2.5 μg/ml for immunofluorescence and 0.25 μg/ml for western blots) (Becton Dickinson), rabbit anti-α-adducin (used at 2 μg/ml for immunofluorescence and 0.2 μg/ml for western blots) (Santa Cruz), rabbit anti-EPB41 (protein 4.1) (used at 1.7 μg/ml for immunofluorescence and 0.17 μg/ml for western blots) (Sigma), rabbit anti-calnexin (Becton Dickinson) (used at 1∶2000).

Techniques: Labeling, Membrane, Mutagenesis, Control, Microscopy

Journal: PLoS ONE

Article Title: The Spectrin Cytoskeleton Is Crucial for Adherent and Invasive Bacterial Pathogenesis

doi: 10.1371/journal.pone.0019940

Figure Lengend Snippet: ( a ) Infections showing spectrin/adducin/p4.1 recruitment to the characteristic actin cups at sites of L. monocytogenes invasion. ( b ) Immunofluorescent image of spectrin RNAi showing inability of L. monocytogenes to invade cells with no detectable spectrin. Arrows indicate internalized bacteria. ( c ) Graphs quantifying invasion efficiency after depletion of spectrin with siRNA as compared to control pool (CP) siRNA treatment. For each treatment, 3 independent experiments were run, for microscopy counts n = 3, error bars show s.e.m, *P<0.0001 for all statistics. ( d ) Initial stages of L. monocytogenes comet tail formation, showing polar spectrin recruitment together with actin. Infection with Listeria Δ actA did not recruit spectrin. Scale bars are 5 µm.

Article Snippet: Antibodies used in this study included a mouse monoclonal anti-β-Spectrin II antibody (used at 2.5 μg/ml for immunofluorescence and 0.25 μg/ml for western blots) (Becton Dickinson), rabbit anti-α-adducin (used at 2 μg/ml for immunofluorescence and 0.2 μg/ml for western blots) (Santa Cruz), rabbit anti-EPB41 (protein 4.1) (used at 1.7 μg/ml for immunofluorescence and 0.17 μg/ml for western blots) (Sigma), rabbit anti-calnexin (Becton Dickinson) (used at 1∶2000).

Techniques: Bacteria, Control, Microscopy, Infection

Journal: PLoS ONE

Article Title: The Spectrin Cytoskeleton Is Crucial for Adherent and Invasive Bacterial Pathogenesis

doi: 10.1371/journal.pone.0019940

Figure Lengend Snippet: Immunolocalization of spectrin to internalized L. monocytogenes at 30 minutes post infection. Scale bars are 5 µm.

Article Snippet: Antibodies used in this study included a mouse monoclonal anti-β-Spectrin II antibody (used at 2.5 μg/ml for immunofluorescence and 0.25 μg/ml for western blots) (Becton Dickinson), rabbit anti-α-adducin (used at 2 μg/ml for immunofluorescence and 0.2 μg/ml for western blots) (Santa Cruz), rabbit anti-EPB41 (protein 4.1) (used at 1.7 μg/ml for immunofluorescence and 0.17 μg/ml for western blots) (Sigma), rabbit anti-calnexin (Becton Dickinson) (used at 1∶2000).

Techniques: Infection