Mab1864

MYO18A-CE antibody was generated by Labcorp by immunizing rabbits with a peptide including the complete 20 residue neoepitope (VKEEDKTLPKPGSPGKEEGA). Equal amounts of protein were loaded on 3–8% Tris-Acetate (MYO18A-CE) or 4–20% Tris-Glycine (TDP-43 and GAPDH) gels and transferred to membranes. The primary antibodies used for the MYO18A-CE experiment were anti-rabbit MYO18A-CE antibody (1:500), anti-rabbit TDP-43 antibody (1:1000, Proteintech, 12892-1-AP), and anti-mouse GAPDH antibody (1:5000, Meridian Life Science, H86504M). For analysis of total HDGFL2 protein levels, equal amounts of protein were loaded in 7% Bis-Tris gels and run with MOPS buffer (Thermo Scientific) and transferred to membranes (Amersham GE Healthcare). Primary antibodies used were rabbit anti-HDGFL2 (1:1000 Sigma HPA 044208), mouse anti-TDP-43 (1:5000 Abcam ab104223) and rat ant-Tubulin (1:5000 Millipore MAB1864). Western blots were developed on a Bio-Rad ChemiDoc and quantified with ImageJ. See supplemental methods section for additional details.

Cells were seeded onto coverslip in a 24-well plate. After indicated treatment, cells were fixed with cold methanol at −20 °C for 5 min and blocked in 3% bovine serum albumin (BSA) at room temperature for 30 min. Cells were incubated with rabbit anti-GFP antibody (1:500, MBL #598), mouse anti-acetylated tubulin (1:2000, Sigma #T7451) or rat anti-α-tubulin (1:2000, Millipore #MAB1864) at 4 °C overnight, washed with PBS followed by incubation with goat AlexaFluor 488-conjugated anti-rabbit (1:500, Invitrogen #A32731), 568-conjugated anti-mouse (1:1000, Invitrogen #A11031) or 647-conjugated anti-rat IgG (1:000, Invitrogen #A21236) for 2 h at room temperature. After washing with PBS containing DAPI, coverslips were washed with deionized water and mounted with FluorSave (EMD Millipore). Images were acquired by fluorescence microscope (Nikon Inverted Microscope Eclipse Ti-U/B, NY, USA). Fluorescence intensity was quantified by ImageJ software (NIH) as described^{12 (link)}.

Cells in chamber slides or 1 cm glass coverslips placed in 12‐well culture plates were cultured for 2 days (Mel270: 140,000 cells; 70,000 cells for all other cell lines). Cells were fixed with ice‐cold methanol for 15 min at −20°C. Subsequent incubations were performed at room temperature for 1 h. Antibodies were diluted in 5% goat serum in PBS. Coverslips were blocked in 5% goat serum (Gibco/Thermo Fisher Scientific) in PBS and then incubated with primary antibody mixes: either rabbit anti‐pericentrin (Abcam ab4448) and mouse anti‐alpha‐tubulin (Sigma‐Aldrich T6199), or rabbit anti‐pericentrin (Abcam ab4448), mouse anti‐Centrin (Millipore, Burlington, MA, USA, 04‐1624), and rat anti‐alpha‐tubulin (Millipore MAB1864). Coverslips were washed with PBS before incubation with secondary antibody mixes whilst protected from light: goat anti‐rabbit Alexa Fluor Plus 555 (Invitrogen, Waltham, MA, USA, A32732), goat anti‐mouse Alexa Fluor Plus 488 (Invitrogen A32723), and, where necessary, goat anti‐rat Alexa Fluor 647 (Invitrogen A21247; all 1/500). Coverslips were washed with PBS before mounting in Mowiol containing 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma‐Aldrich) at 1 μg/ml. Slides were stored at 4 °C, protected from light.

Primary antibodies used were rabbit anti-Fltp1 (Pineda, Berlin), rabbit anti-Fltp116-1 (Pineda, Berlin), mouse anti-ZO-1 (33–9100: Invitrogen, Carlsbad, CA), mouse anti-α-Tubulin (T6199; Sigma), mouse anti-acetylated Tubulin (T7451; Sigma), mouse anti-γ-Tubulin (ab11316; Abcam), rabbit anti-β-Catenin (C2206; Sigma), chicken anti-GFP (GFP-1020; Aves Labs), rat anti-tyrosinated Tubulin (MAB1864; Millipore), rabbit anti-pericentrin (PRB-432C; Covance), rabbit anti-Vangl1 (HPA025235; Sigma), rabbit anti-Dvl2 (3216; Cell Signaling), and Alexa Fluor 546 Phalloidin (A22283; Invitrogen). Immunostainings were performed as described in the Supplemental ‘Materials and methods’.

MEFs were prepared from e13.5 control and Tie2Cre;Ino80 fl/fl embryos. Cells were grown in DMEM with 10% FBS and infected with either adenovirus expressing Cre recombinase (Ad5-CMV-Cre-GFP) or empty vector (Ad5-CMV-GFP) after 4 passages. After 48 h, GFP expression was measured at >95%. After 72 h after infection, protein was collected for western analysis using anti-Ino80 antibody (Abcam ab105451) and anti-tubulin antibody (Millipore MAB1864). Uncropped western images shown in Supplemental Fig. 7.

Larvae were dissected as previously described (Collins et al., 2017 (link); Camuglia et al., 2018 (link)). Briefly, larvae were dissected in ice-cold 1,4-piperazinediethanesulfonic acid (PIPES) dissection buffer containing 100 mM PIPES (P6757; Sigma- Aldrich), 115 mM d-sucrose (BP220-1; Fisher Scientific), 5 mM trehalose (182550250; Acros Organics), 10 mM sodium bicarbonate (BP328-500; Fisher Scientific), 75 mM potassium chloride (P333- 500; Fisher Scientific), 4 mM magnesium chloride (M1028; Sigma- Aldrich), and 1 mM ethylene glycol tetraacetic acid (28-071-G; Fisher Scientific) and then fixed with 4% Formaldehyde in PIPES buffer (BP531-500; Fisher Scientific).
Antibodies for larva filet staining were used at the following final dilutions: rat anti- tyrosinated tubulin, 1:400 (MAB1864; Millipore), mouse anti-myospheroid (β-integrin), 1:100 (CF.6G11, Developmental Studies Hybridoma Bank). Larval NMJs were labeled with Alexa-Fluor 647-conjugated goat anti-HRP, 1:500 (123-605-021,Jackson ImmunoResearch Laboratories). We used Alexa Fluor 488- and Alexa Fluor 555- conjugated fluorescent secondary antibodies (1:400; Life Technologies), and Hoechst 33342 (1 μg/ml, ThermoFisher). Larvae were mounted in ProLong Gold (P36930; Life Technologies).
All images were acquired on a Zeiss 700 LSM using an Apochromat 40×/1.4 numerical aperture (NA) objective.