Mouse anti α tubulin clone dm1a

Briefly, 2 × 10⁵ cells were seeded in µ-slide 8-well chambered coverslips (Ibidi, Gräfelfing, Germany) and incubated in culturing medium containing either DMSO or mebendazole for 24 h. In addition, 5 µm cryosections of tumor samples of the animal studies that have been air-dried for 10 min were used. Cells or tissue sections were fixed in 4% paraformaldehyde/PBS for 15 min, permeabilized with 0.5% Triton X-100 for 1 h and then blocked in PBST containing 3% BSA and 0.1% glycine for 10 min. Slides were then incubated overnight at 4 °C with mouse anti-α-tubulin (clone DM1A) (Sigma-Aldrich), Ki67 (Abcam, Cambridge, UK) and cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA) antibodies in 5% BSA/PBS at dilutions of 1:100, 1:250, and 1:100, respectively. The next day, following serial washing steps, slides were incubated with Alexa Fluor 555-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen) in a dilution of 1:250 in 5% BSA and 0.1% Triton X-100. Slides were counterstained with 1 µg/mL Hoechst 33342 (Invitrogen) at room temperature in the dark for 1 h. Images of tubulin staining were captured with the 40× objective of the Axiovert 200M microscope equipped with an AxioCam MRm (Zeiss, Jena, Germany) and abnormal spindles were counted by ImageJ. Ki67 and caspase-3 stainings were captured with the EVOS M7000 microscope (Invitrogen).

Cells were grown to 70–80 % confluence in 10 cm dishes, washed with 1XPBS and harvested in 1xSDS buffer. Lysates were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. Chemiluminescence was used for detection using the western HRP substrate reagent (Millipore) and visualized on a GE Image Quant LAS4010 Imaging System. Blots were stripped in 0.1 M Glycine, 0.5 % SDS (pH2.5) for 1 h. Blots were re-blocked and probed with mouse-anti-α-tubulin (clone DM1A, T9026, Sigma) to assess loading. Chemiluminescence and visualization is performed as described previously. Pan-FMNL2 antiserum was raised in chicken (Cedarlane) using the isolated FMNL2-FH2 protein (codons 599–1045) expressed in E.coli and purified as previously described [11 (link)]. All FMNL2 antisera were affinity purified using standard protocols [40 (link)]. Affinity purified anti-FMNL3 antibody was described previously [41 (link)].

Protein samples were isolated from the PFC tissue blocks and separated on 8% SDS polyacrylamide gels. The primary antibodies are as follows: mouse anti-Nav1.2, clone K69/3, 1:200 (Neuromab, Davis, CA, United States); rabbit anti-Nav1.6, 1:200; rabbit anti-SK1, #APC-039, 1:200; rabbit anti-SK2, #APC-028, 1:200; rabbit anti-SK3 N-term, #APC-025, 1:200 (Alomone Labs, Jerusalem, Israel); mouse anti-α-tubulin, clone DM1A, 1:1000 (Sigma-Aldrich, St. Louis, MO, United States). We used 3 and 4 animals for control and MIA-SI groups, respectively. And experiments were repeated for 3–4 times. The integrated density of samples on blots were analyzed using ImageJ (National Institutes of Health, United States). For each blot, we normalized the integrated density of each sample to β-tubulin and then normalized this ratio to the mean ratio of samples in control groups. Then we averaged the normalized density of each sample, and performed statistical analysis (Student’s t-test).

For quantification of ERK and pERK levels, 50 ventral ganglia from late third instar larvae were dissected in ice-cold fixation solution (4% paraformaldehyde in PBS) to minimize changes in phosphorylation status during handling, fixed for 10 min on ice and washed three times for 20 min in PBS. Following homogenization and sonication, protein lysates were analyzed by western blotting with the following antibodies: monoclonal rabbit anti-Phospho-p44/42 (ERK1/2, Thr202/Tyr204; clone D13.14.4E, 1:2000; Cell Signaling), monoclonal rabbit anti-p44/42 (ERK1/2; clone 37F5, 1:1000; Cell Signaling) and mouse anti-α-tubulin (clone DM1a, 1:10,000; Sigma-Aldrich). Sheep anti-mouse-HRP (1:10,000; GE Healthcare) and donkey anti-rabbit-HRP (1:10,000; GE Healthcare) were used as secondary antibodies. Western blots were scanned with an Intas ChemoCam imager, and intensities of protein bands were measured with the ImageJ 1.48a software (NIH, Bethesda, MD, USA). The intensity of the α-tubulin band was used for normalization.

The following antibodies were used for immunofluorescence: mouse anti α-tubulin (clone DM1A, Sigma-Aldrich) 1:1000; rabbit anti-Msps rb2219 (this study) 1:1000; rabbit anti-Cnn (Bettencourt-Dias et al., 2005 (link)) 1:2000; chicken anti-Dplp (Rodrigues-Martins et al., 2007 (link)) 1:1000; rabbit anti-mCherry (Abcam ab167453) 1:2000; mouse anti-LAMP1 human (clone H4A3, DSHB) 1:500; anti γ-tubulin (clone GTU-88, Sigma-Aldrich) 1:1000, rabbit anti-Rab5 antibody - Drosophila Early Endosome Marker (Abcam ab31261) 1:100; Recombinant rabbit monoclonal Anti-hRab5 antibody (Abcam ab218624) 1:1000
A rabbit anti-Msps was generated against the 1350-1785 amino acid fragment of Msps. DNA encoding the fragment was amplified by PCR from cDNA (Table S1), cloned in the pDONR221 entry vector, and recombined with the pDEST17 destination vector to generate a recombinant DNA encoding a 6xHis N-terminally tagged fusion fragment. The 6xHis fragment was expressed in bacteria, affinity purified on Ni-NTA Agarose beads (Qiagen) and used to for rabbit immunizations (Harlan UK). The final bleed was used for immunostainings and western blots.

The following primary antibodies were used: rabbit anti-Pan-cadherin (C3678, Sigma-Aldrich, St. Louis, MO) that recognizes the conserved C-terminal domain of classic cadherins, mouse anti-N-cadherin (clone 32) and anti-E-cadherin (clone 36), both from BD Biosciences (Franklin Lakes, New Jersey, USA), rabbit anti-β-catenin (Invitrogen-Molecular Probes, Carlsbad, CA), mouse anti-active-β-catenin (clone 8E7, Millipore, Billerica, MA, USA), mouse anti-lamin A∖C (BD Biosciences), and mouse anti-α-tubulin (clone DM1a, Sigma-Aldrich). Secondary antibodies were Alexa Fluor™ 488 goat anti-rabbit IgG, Alexa Fluor™ 546 rabbit anti-mouse IgG (Invitrogen, Life Technologies, Brazil, São Paulo, SP, Brazil), and peroxidase-conjugated goat anti-rabbit and rabbit anti-mouse (Promega, Madison, WI). DAPI dihydrochloride (Invitrogen) was used for nuclear staining. The γ-secretase activity inhibitor Dapt (N-N[-(3,5-Difluorophenacetyl-l-alanyl)]-S-phenylglycine-t-butyl-ester) was from Merck Biosciences (Darmstadt, Germany). Nuclear and cytoplasmic fractions were extracted using NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL).