Donkey anti goat sc 2020

[³H]CGP12177 (specific activity 41.6 Ci/mmol) was purchased from Perkin Elmer Health Sciences (Toronto, ON, Canada). Antibodies against rat β₁-AR (Ab3546) were purchased from AbCam (Cambridge, MA, USA). Antibodies against β₂-AR (SC-570), β₃-AR (SC-1473), and GAPDH (SC-32233) were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Secondary horseradish peroxidase conjugated IgG antibodies goat anti-rabbit (SC-2004), donkey anti-goat (SC-2020), and donkey anti-mouse (SC-2314) were also purchased from Santa Cruz Biotechnology.

For immunoblotting, the following antibodies were used: rabbit anti-IKK1/2 (sc-7607), rabbit anti-Mac2/galectin-3 (sc-20157), rabbit anti-Erk2 (sc-154), HRP-conjugated goat anti-mouse (sc-2005), goat anti-rabbit (sc-2004), and donkey anti-goat (sc-2020) from Santa Cruz Biotechnology (Heidelberg, Germany). Mouse anti-β-amyloid (6E10, sig-39320) from Covance (Munich, Germany) and goat anti-Lcn2 (AF1857) from R&D (Minneapolis, MN, USA). For proper quantitative analysis, donkey anti-mouse and donkey anti-rabbit IRDye 680LT-conjugated secondary antibodies (#926-68022 and #926-68023) as well as donkey anti-mouse and anti-rabbit IRDye 800CW-conjugated secondary antibodies (#926-32212 and #926-32213) from LI-COR^® were used:
For immunofluorescent staining, the following antibodies were used: goat anti-GFAP and rabbit anti-Mac2/galectin-3 (sc-20157) from Santa Cruz Biotechnology (Heidelberg, Germany), chicken anti-GFAP (ab6476) from Abcam (Berlin, Germany), rabbit anti-Iba1 (019-19741) from WAKO (Neuss, Germany), and mouse anti-β-amyloid (4G8, sig-39320) from Covance (Munich, Germany). Alexa Fluor labeled secondary antibodies (488, 568/594, and 647) were obtained from Invitrogen (Waltham, MA USA) and DAPI was purchased from MERCK (Darmstadt, Germany).

Western blots were performed essentially as described in [32 (link)] with the following modifications: a protease inhibitor cocktail (Roche, 1:25) was used and no additional dithiothreitol was added to the sample mix including loading dye. Aliquots of cell lysates were further applied for Western blot analysis as published previously [15 (link)]. The following proteins were detected by specific antibodies: OPN (M66105M, Meridian Life Science, Memphis, TN, USA), PI3Kinase p110 β (#3011), pPDK1 (#3438), pAkt (#13038P), pmTOR (#5536) (Cell signaling technology, Frankfurt, Germany), and β-actin (sc1615, Santa Cruz, Heidelberg, Germany), which served as internal loading control. The respective horseradish-peroxidase-conjugated secondary antibodies were donkey anti-goat (sc2020), goat anti-mouse (sc2055) (Santa Cruz), and goat anti-rabbit (#7074, Cell signaling technology).

Immunoblotting was done as described in (Yosifov et al. 2007 (link)), but a proteinase inhibitor cocktail (Roche, 25x) was used and additional dithiothreitol was not included to the sample mix and loading dye. Aliquots of the cell lysates were further analyzed as described before (Kovacheva et al. 2014 (link)). Specific antibodies detected the following proteins: integrin β3 (sc-46655) and β-actin (sc1615) (Santa Cruz, Heidelberg, Germany). Beta-actin served as internal loading control. The respective horseradish-peroxidase-conjugated secondary antibodies were goat anti-mouse (sc 2055) and donkey anti-goat (sc2020) (Santa Cruz, Heidelberg, Germany).

Whole organoid lysates were prepared with a lysis buffer containing 8 M urea. 15 μg of proteins from each CI994-treated and untreated condition were separated on 4–12% Bis-Tris NuPage precast gels (Life Technologies) and transferred to PVDF blotting membranes (Roche Applied Science). Western blotting was performed as described previously21 (link). Antibodies used were: goat anti-Reg3β (AF1996, R&D Systems), goat anti-SIS (sc-27603, Santa Cruz Biotechnology), mouse anti-actin (MAB-1501, EMD Millipore), donkey anti-goat (sc-2020, Santa Cruz Biotechnology) and goat anti-mouse (sc-2005, Santa Cruz Biotechnology). Immune complexes were detected with Amersham ECL™ Western blotting detection reagents (GE Healthcare), according to the manufacturer’s instructions.

The following antibodies were used for blotting: polyclonal goat anti-mouse κ LC (1050–01, SouthernBiotech); mouse anti-GAPDH (MAB374, Millipore); mouse anti-Hsc70 (B-6) (sc-7298, Santa Cruz Biotechnology), mouse anti-VCP/p97 (ab11433, abcam, Cambridge, MA), mouse anti-20S proteasome subunit alpha (C8) (PW8110, Biomol, Hamburg, Germany), rabbit anti-PSMC2 (HPA019238, Sigma-Aldrich), mouse anti-actin (A5441, Sigma-Aldrich), rabbit anti-PDIA6 (NBP157999, Novus Biologicals). HRP-conjugated antibodies were used at 1:10,000 dilution and include goat anti-rabbit (sc-2054), donkey anti-goat (sc-2020), and goat anti-mouse (sc-2031) all purchased from Santa Cruz Biotechnology. The rabbit anti-ERdj3 antibody was produced in our lab (Shen and Hendershot, 2005 (link)). IRdye^® secondary antibodies (LI-COR Biosciences, all 1:20,000): goat anti-mouse IgG (925–32210), goat anti-rabbit IgG (925–68071), and donkey anti-goat IgG (926–68074).