Goat anti mouse igg

OVCAR-3 and SKOV-3 cells were maintained in complete RPMI-1640 in the absence or presence of 30 μM PD 98,059 or 10⁻⁷ M ICI 182,780 (Selleck Chemicals). Nucleoproteins or whole cell lysates were prepared and separated on discontinuous SDS-PAGE, then transferred by electroblotting to PVDF membrane (Millipore, Bedford, MA, USA), as we have previously described [12 (link), 19 (link)]. Membranes were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween, and then incubated with selected antibodies overnight: PCNA (GTX100539, GeneTex Inc. CA, USA), Lamin B (GTX103292, GeneTex Inc.), integrin αv (SC9969, Santa Cruz Inc., Santa Cruz, CA, USA ), integrin β3 (SC6627, Santa Cruz), phospho-ERK1/2 (4377S, Cell Signaling Technology, Danvers, MA, USA), total-ERα (8644S, Cell Signaling Technology), ERα-pS118(2511p, Cell Signaling Technology), ERα-pS167 (5587p, Cell Signaling Technology). Horseradish peroxidase (HRP)-conjugated secondary antibodies were either goat anti-rabbit IgG or goat anti-mouse IgG (1:1000, Dako, Carpenteria, CA, USA), depending on the origin of the primary antibody. Immunoreactive proteins were detected by chemiluminescence.

Whole cell extracts were prepared in RIPA cell lysis buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate) containing protease and phosphatase inhibitors (Roche Life Science, Penzberg, Germany). The samples were separated by sodium dodecyl sulfate-13% polyacrylamide gel electrophoresis (SDS-PAGE), and then blotted onto a PVDF membrane (Amersham, GE Healthcare Life Sciences, Little Chalfont, UK). Immunoblot was performed as previously described^{65 (link)} using antibodies from following sources: anti-p53 (DO-1; Santa Cruz Biotechnology, Dallas, TX, USA), anti-p21 (F-5; Santa Cruz Biotechnology), anti-p16 (BD Biosciences), anti-human retinoblastoma protein (pRb; BD Biosciences), anti-phospho-histone H3 Ser10 (p-H3, Cell Signaling Technology, Danvers, MA, USA), anti-histone H3 (Merck Millipore, Darmstadt, Germany) and anti-β-actin (Sigma-Aldrich). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin (Ig) G (AgriSera AB, Vännäs, Sweden) or goat anti-mouse IgG (Dako, Glostrup, Denmark) was used as a secondary antibody. Immunoreactive bands were visualized using a Clarity Western ECL substrate kit (Bio-Rad, Hercules, CA, USA) on a LAS-3000 luminescent image analyser (GE Healthcare).

Rabbit polyclonal antibody used was anti-SEPT7 (IBL 18991, 1:500). Mouse monoclonal antibody used was GAPDH (AbCam ab8245, 1:500). Horseradish peroxidase-conjugated secondary antibodies used were goat anti-rabbit-IgG (Dako P0448, 1:2000) or goat anti-mouse-IgG (Dako P0260, 1:2000). F-actin was labelled with Alexa Fluor 488- or Alexa Fluor 555-conjugated phalloidin (Molecular Probes A12379 or A34055, 1:100).

Donor cells were permeabilized with 0.1% saponin and 5% FBS for 20 min. After fixation, co-cultures were washed with 100 mM glycine in PBS and permeabilized/blocked in a PBS buffer containing 0.02% Triton X-100, 0.1% BSA, and 0.05% Tween-20, along with 10% goat serum (Sigma-Aldrich) and 1% goat anti-mouse IgG (Dako). The following primary antibodies were used: mouse anti-KDEL (marker for endoplasmic reticulum and cis-Golgi network, 1:400; Enzo Life Sciences), mouse anti-SV2 (Synaptic vesicle glycoprotein 2a, marker for the synapse and synaptic vesicles, 1:500; deposited by K.M. Buckley to the Developmental Studies Hybridoma Bank, The University of Iowa), mouse anti-TSG101 (tumor susceptibility gene 101, marker for multi-vesicular bodies (MSBs) and other endosomal compartments, 1:400; Thermo Pierce), mouse anti-VAMP2/synaptobrevin (Vesicle-associated membrane protein 2, marker for the synapse and synaptic vesicles, 1:100; Synaptic Systems), and mouse anti-LAMP2 (Lysosomal associated membrane protein 2, marker for lysosomes/endosomes, 1:100; Southern Biotechnology). Incubation with the indicated primary antibody (overnight, 4°C) was followed by incubation with a secondary antibody conjugated with either Alexa Fluor 405 or Alexa Fluor 488 (Invitrogen) (75 min for donor cells only or 45 min for co-culture, RT). Finally, slides were mounted with ProLong containing DAPI.

Two of the identified immune-related proteins, Cyclophilin A (CypA) and Cofilin 1 (Cof1), that were included in the list of proteins contributing to the separation of the TDI-treated group and AOO-treated group in the PCA were verified via Western blotting in B lymphocytes obtained from a new, independent set of similarly treated TDI- and AOO-treated mice (n = 10/group). Briefly, proteins were loaded and separated on 4–12% Bis/Tris Midi-gels (Invitrogen, Merelbeke, Belgium) and subsequently transferred to a PVDF membrane (iBlot, Gel Transfer Stack, Invitrogen). Membranes were blocked (1–2 h, 5% blocking agent, GE Healthcare) and incubated overnight with primary antibody (LSP-1: 1/1000, goat Ab, Santa Cruz Biotechnology; CypA: 1/1000, mouse Ab, Santa Cruz Biotechnology; GAPDH, internal standard, 1/200000, mouse Ab, Dako). Following secondary Ab incubation (LSP-1: 1/50000, donkey anti-goat IgG, Santa Cruz Biotechnology; Cof1 and GAPDH: 1/100000, goat anti mouse IgG, Dako) protein bands were visualized using chemiluminescence detection (Supersignal West Dura, Thermo Scientific) on ECL hyperfilm (GE Healthcare). The protein bands were semiquantitatively evaluated by densitometry (ImageQuant TL v2009, GE Healthcare).

For protein isolation snap-frozen tissue of iBAT and muscle were homogenized in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 0.1% w/vol SDS, 0.5% w/vol sodium deoxycholate, 1% vol/vol Nonidet P40, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM NaVO₄ and 10 mM NaF) and freshly added protease inhibitors (Roche Diagnostics GmbH, Germany). Protein concentrations were determined using a bicinchoninic acid assay (BCA, Sigma, Germany). For immunological detection, 20 µg of iBAT protein were separated on a 12% SDS polyacrylamide gel (BioRad Laboratories, Germany) and transferred onto a polyvinylidene fluoride membrane (Merck Millipore, Germany). The membranes were probed with a rabbit anti-UCP1 antibody (customized rabbit antibody raised against UCP1^{63 (link)}, or a mouse anti-total OxPhos antibody cocktail (#45-8099, Invitrogen Germany), followed by a peroxidase-conjugated secondary antibody (goat anti-rabbit-IgG, #P0448, DAKO, Denmark or goat anti-mouse-IgG, #P0447, DAKO, Denmark). The antigens were visualized using an ECL Plus system (Chemi Doc Touch, BioRad). Band intensity was quantified using Image Lab™ software (BioRad) and data were normalized to total protein load.

Proteins were extracted using RIPA buffer supplemented with 1 mM PMSF and 20–50 µg of cell lysates were separated with SDS–PAGE as described [29 (link)]. The primary antibodies rabbit anti-Smg6/Est1A (1:1500; Abcam, Berlin, Germany), mouse anti-GAPDH (1:20,000; Sigma-Aldrich, Taufkirchen, Germany), mouse anti-Actin (1:20,000; Sigma-Aldrich), and secondary antibodies HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:10,000; DAKO, Frankfurt am Main, Germany), were used.