Anti α actinin

The following antibodies were used at the indicated dilutions: anti-PKD1 (sc-935; 1/500) and anti-α-actinin (1/5000) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-phospho-PKD1 (1/1000), anti-cleaved PARP (1/1000), anti-ERα (1/2000), anti-phospho-S118-ERα (1/2000) and anti-phospho-S167-ERα (1/2000) were from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated secondary antibodies used were goat anti-rabbit IgG (1/2000; Dako, Glostrup, Denmark) and goat anti-mouse IgG (1/5000; Rockland, Gilbertsville, PA, USA). PKD1-targeting (sc-36245) and control non-targeting (sc-37007) siRNAs were purchased from Santa Cruz Biotechnology. 17β-estradiol, ICI 182,780, MTT and all other biochemicals were from Sigma-Aldrich (St. Louis, MO, USA).

Plasmids encoding Myc-tagged IRF5 isoforms were a kind gift of Dr. Frank Neipel (Virologisches Institut - Klinische und Molekulare Virologie, Erlangen, Germany). Plasmids encoding Xpress-TRIM21 and GST-TRIM21 PRY/SPRY domain were a gift from Dr. David Rhodes (Cambridge Institute for Medical Research, Cambridge, UK). HA-ubiquitin wild type and mutants were a gift from Dr. James Burrows (Centre for Cancer Research and Cell Biology, Belfast, UK). Plasmids encoding FLAG-tagged IRF5 full length and deletion mutants were described previously [24] (link). Myc-MyD88 construct was a kind gift from Dr. Alberto Mantovani (Istituto Clinico Humanitas, Milan, Italy). pGL3-IFNA4 luciferase and pGL4-TK-Renilla were a kind gift from Dr. John Hiscott (Lady Davis Institute, Montreal, Canada) and Dr. Kate Fitzgerald (UMASS, Massachusetts, USA), respectively. Plasmids encoding shRNA targeting TRIM21 and scrambled negative control were described previously [16] (link). TLR ligands were purchased from InvivoGen (California, USA). Primary antibodies used were anti-FLAG (Sigma), anti-c-Myc and anti-β-Actin (Abcam), anti-GST (GE Healthcare), anti-Xpress (Invitrogen), anti-IRF5 (Cell Signaling) and anti-α-actinin, anti-HA and anti-TRIM21 (Santa Cruz).

The mice were sacrificed by a lethal injection of pentobarbital (200 mg/kg). Mouse embryos were extracted at E16. The tibialis anterior, triangularis sterni, soleus, and gastrocnemius muscles were excised and fixed in 4% PFA or cold methanol. Muscles were stripped of connective tissues and either used for the preparation of teased fibers or stained in a whole-mount procedure. Nonspecific staining was blocked with 2% bovine serum albumin and 2% goat serum in PBS supplemented with 0.5% Triton-X100 before overnight incubation with primary antibodies. The muscles were stained using fluorescently labeled BTX (catalog no. B35451, Life Technologies) and primary antibodies against anti-liprin-α-1 (catalog no. 115098, GeneTex), anti-α-actinin (catalog no. sc-17829, Santa Cruz Biotechnology), anti-ryr (catalog no. ab2868, Abcam), anti-myomesin (catalog no. ab111433, Abcam), anti-α-dystrobrevin-1 (catalog no. 610766, BD Transduction Laboratories), anti-neurofilament (catalog no. 2H3, Developmental Studies Hybridoma Bank), and anti-synaptophysin (catalog no. 101 004, Synaptic Systems). For cryosectioning tibialis anterior muscles were flash-frozen in isopentane cooled with liquid nitrogen and sectioned at 10–20 µm in a cryostat. Cryosections were fixed and stained as described above.

Proteins from lung tumors of SPC/c-Myc-transgenic mice and/or non-transgenic animals were extracted and quantified as previously reported [7 (link)]. Seventy-five or 100 μg (50 μg in case of HEK293T over-expressing c-MYC) of total protein extracts were separated on 12.0% SDS-polyacrylamide gel and blotted onto PVDF membranes in 25 mM Tris and 190 mM glycine at 4 °C for 2 h at 350 mA or using the semi-dry iBlot transfer system (InVitrogen, Life Technologies). Specific antibodies were used in dilution given in parenthesis for the detection of anti-Traf4 (1:100), anti-hepsin (1:100), anti-Alk1 (= Acvrl1) (1:100), anti-Igfbp6 (1:100), anti-c-MYC (1:1000) and anti-α-Actinin (1:8000) purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany); anti-claudin2 (1:500) purchased from Zymed Laboratories, Inc. (San Francisco, CA, USA) and anti-Adam19 (1:1000) purchased from Cedarlane Laboratories (Burlington, Ontario, Canada). Antigen-antibody complexes were visualized using the ECL detection system NEN Life Science Products (PerkinElmer Life Science, Rodgau-Juegesheim, Germany) or ECL Select kit (GE-Healthcare, Milan, Italy) as recommended by the manufacturer and recorded with the Kodak IS 440 CF (Kodak, Biostep, Jahnsdorf, Germany) and a ChemiDoc XRS+ (BioRad, Milan, Italy).

The primary antibodies used were anti-c-Myc (Y69, ab32072) and anti-Oct4 (1:500, ab19857), obtained from Abcam (Abcam, Cambridge, MA, USA). Anti-β-actin (1:3000, sc-47778), anti-α-tubulin (1:2000, c-5286), anti-HDAC-1 (1:500, SC, #81598), and anti-α-actinin (1:500, SC, 17829) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All of the following antibodies were purchased from Cell Signaling (Danvers, MA, USA): anti-Sox2 (1:1000, D6D9), anti-KLF4 (1:500, CST, #4038S), anti-STAT3 (1:1000, CST, #124H6), anti-pSTAT3^Y705 (1:2000, CST, #9145), anti-pALKY1604 (1:500, CST, #3341S), and anti-ALK (1:1000, CST, #3633). The secondary antibodies used were HRP-conjugated anti-mouse (1:2000, CST, #7076) and anti-rabbit (1:2000, CST, #7074). Puromycin, doxorubicin, crizotinib, and etoposide were all purchased from Sigma-Aldrich (Oakville, ON, Canada). All treatments were performed following the manufacturer’s instructions.