Sheep anti mouse igg hrp

Primary antibodies used included mouse monoclonal anti-pTyrosine, clone PY20 (ExAlpha, Cat # X1021, Lot # 12175) and monoclonal rabbit EGF Receptor (D38B1) XP^® antibody (Cell Signaling, Danvers, MA, Cat # 4267S, Lot # 8). The strong pTyr signal in the post-kinase reaction vs the pre-kinase reaction along with the 1D linear WB response validated the anti-pTyr antibody for this study. MS confirmed that the post-kinase ALK48 contained pTyr. Secondary antibodies used included sheep anti-mouse IgG-HRP (GE Healthcare, Cat# NA931, RRID:AB_772210, Lot # 9729340) and sheep anti-rabbit IgG-HRP (GE Healthcare, Cat# NA934, RRID:AB_772206, Lot # 9495175).

Protein extracts were separated on 7.5 or 10% Mini-PROTEIN TGX Precast Gels (Bio-Rad) and then transferred onto nitrocellulose membranes with the Trans-Blot Turbo Transfer System (Bio-Rad). The following primary antibodies and dilutions were used: rabbit anti-PRDM9 polyclonal (Grey et al. 2017 (link)), 1:500; rabbit anti-CTCF monoclonal (Cell Signaling Technology, D31H2), 1:2000; rabbit anti-CXXC1 polyclonal (Millipore, ABE211), 1:5000; rat anti-tubulin α monoclonal (Abcam, ab6161), 1:3000; rabbit anti-PIH1D1 polyclonal (Proteintech, AP19427), 1:5000; rabbit anti-CEP70 polyclonal (Abnova, PAB17658), 1:1000; mouse anti-MCRS1 polyclonal (Abnova, H00010445-B01P), 1:250; anti-Gal4 AD (Millipore, 06-283), 1:3000; and anti-Gal4 BD (Sigma, G3042), 1:2000. Signals were detected with the horseradish peroxidase (HRP)-conjugated secondary antibodies, donkey anti-rabbit IgG HRP (Jackson ImmunoResearch Laboratories), goat anti-rat IgG HRP (GE Healthcare), and sheep anti-mouse IgG HRP (GE Healthcare), and then visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). ImageJ 1.49m was used to quantify signal intensities of western blot images captured by ChemiDoc™ XRS+ Imager with the Image Lab 5.1 software.

The microdissected growth plate obtained as described above was homogenized in 50 µl of Laemmli sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% (v/v) glycerol, 0.01% bromophenol blue) and the protein content was determined with the BCA Protein Assay (Pierce). Samples were then reduced in 5% β-mercaptoethanol, separated by 7% SDS–PAGE and blotted on a polyvinylidenedifluoride membrane (PVDF, Hybond). Primary antibodies used in this study were: anti-β-catenin, anti-cyclin D1, and anti-p107 (sc-7199, sc-753, and sc-318 respectively, Santa Cruz Biotechnology, Inc.), anti-p130 (610261, BD Transduction Laboratories™), anti-phospho-β-catenin (Ser33/37/Thr41, 9561, Cell Signaling Technology), and anti-β-actin (A5316, Sigma–Aldrich). The following secondary HRP-conjugated antibodies were used: donkey anti-rabbit IgG-HRP (sc-2077, Santa Cruz Biotechnology, Inc.) and sheep anti-mouse IgG-HRP (NA931, GE Healthcare). Four percent milk powder in 1× TBS-T (20 mM Tris–HCl, pH 7.5, 0.5 mM NaCl, and 0.1% (v/v) Tween-20) was used for blocking and for diluting antibodies. Chemiluminescence detection was achieved with the ECL Plus Western blotting detection reagents (GE Healthcare) and autoradiography films (Hyperfilm, GE Healthcare). Films were digitalized by VersaDoc™ Imaging System (Bio-Rad) and densitometric analysis was performed by Quantity One software (Bio-Rad).

The following antibodies were used in this study: anti-L3MBTL2 (Active Motif, 39,569), anti-E2F6 (Diagenode, C15410314), anti-RBPJ (Cell signaling, 5313), rabbit IgG (Diagenode, C15410206), mouse IgG (Santa Cruz, sc2025), anti-Flag (Merck, F3165, F4042), anti-Flag HRP (Merck, A8592), anti-GFP (Merck/Roche, 11,814,460,001), anti-VP16 (Santa Cruz, sc-7545), anti-GAPDH (Abcam, ab8245), anti-TBP (Abcam, ab818), anti-GST (kind gift from Dr. M.L. Schmitz), anti-SUMO2/3 (Abcam, ab81371), anti-VINCULIN (Abcam, ab130007), anti-mouse IgG-HRP (Cell signaling, 7076 or Amersham NXA931), anti-rabbit IgG HRP (Cell signaling, 7074), anti-rat IgG HRP (Jackson ImmunoResearch, 112-035-072), sheep anti-mouse IgG HRP (GE Healthcare, NA931V), anti-Flag (M2) conjugated agarose beads (Merck, A2220), Hemaglutinin (HA) (Covance, MMS-101P).

Protein extracts and nuclear extracts were obtained from H9C2 cells and MDA-MB-231 cells. Western blotting was performed as previously described [27 (link),28 (link),29 (link)]. For western blotting, anti-cleaved caspase-3 antibody, anti-cleaved caspase-9 antibody (Abcam, Cambridge, UK), anti-p53 antibody, phospho-p53 (Ser15) antibody (Cell Signaling Technology, Danvers, MA, USA), and anti α-tubulin monoclonal antibody (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) were used as primary antibodies, and goat anti-rabbit IgG–HRP (MBL, Aichi, Japan) and sheep anti-mouse IgG-HRP (GE Healthcare, Chicago, IL, USA) were used as secondary antibodies.

Cell lysates from OF at various time points before and during adipogenesis were obtained by addition of lysis buffer as previously described [13 (link)]. The culture supernatants from the same time points were also collected and concentrated using spin columns (Merck Millipore, Watford, Hertfordshire, UK) to produce an 80-fold concentration. Lysates and concentrated supernatants were separated using SDS-PAGE as previously described [13 (link)]. Briefly, proteins were extracted, at various time points, in Laemmli buffer containing 1 mM phenylmethylsulphonyl fluoride. Samples were separated by 10% SDS-PAGE and then the gel electroblotted onto PVDF membrane. The blots were probed using antibodies to the full length TSHR (2C11, Santa Cruz Biotechnology, Heidelberg, Germany) and TSHRv antibody, at dilutions of 1:200 and 1:50 respectively at 4 °C overnight. This was followed by a sheep anti-mouse IgG-HRP (1:5000, room temperature for 1 h, GE Healthcare) or donkey anti-rabbit IgG-HRP conjugate (1:5000, room temperature for 1 h, GE Healthcare) and then visualised by enhanced chemiluminescence using ECL Plus (Amersham Pharmacia Biotech, Buckinghamshire, UK). They were then stripped and reprobed with antibodies to housekeeping protein, actin at dilution of 1:1000 4 °C overnight with secondary anti-rabbit as above.