Rabbit anti gapdh hrp

Cells were lysed in RIPA buffer (MilliporeSigma) supplemented with proteinase (ThermoFisher) and phosphatase (PhosSTOP, Roche Diagnostics, Indianapolis, IN) inhibitors. The concentration of cell lysates was determined using bicinchoninic acid assay (BCA) (ThermoFisher). Samples were loaded onto 4–20% Mini-Protean TGX Precast Gels (Bio-Rad) and then transferred to PVDF membranes (Bio-Rad) using Trans-Blot Turbo System (Bio-Rad). Membranes were incubated with following primary antibodies: mouse anti-total tau A0024 (DAKO; 1:1000, # A0024), mouse anti-synapsin (ThermoFisher; 1:1000, MA531919), rabbit anti-Gapdh-HRP (Cell Signaling; 1:2000, #8884), rabbit anti-Rgp antibody CAB101 (1:1000) [1 (link)], and rabbit anti-Kgp antibody CAB102 (1:1000) [1 (link)]. All secondary antibodies were horseradish protein (HRP) conjugated (Vector; 1:5000 or 1:10000). Blots were imaged by chemiluminescent detection using Supersignal West Fento (ThermoFisher) and the ChemiDoc imaging system. Quantification was performed with Image Lab 6.0.1 software (Bio-Rad) by dividing the integrated OD of all bands of interest by the OD of GAPDH detected on the same blot.

Lysates of hpRPE cells were generated using RIPA buffer (Sigma-Aldrich, Munich, Germany, #R0278) with protease and phosphatase inhibitors (1:100, Sigma-Aldrich, #P8340). Samples were dissolved in reducing Laemmli sample buffer, denatured (95 °C, 10 min) and separated in a 12% SDS-PAGE and transferred on to an activated PVDF membrane using a wet blotting system. Membranes were blocked (1 h, 5% bovine serum albumin (BSA)/PBS-T) and incubated with the primary antibodies (overnight, 5% BSA/PBS-T): Rabbit anti-GAPDH-HRP (1:1000, Cell Signaling Technology, Beverly, MA, USA, #3683), mouse anti-C3 (1:100, Progen, Heidelberg, Germany, #61019), goat anti-CFI (1:250, Quidel, San Diego, CA, USA, #A313), mouse anti-C3a (1:50, Hycult, Uden, Netherlands, #HM2074) and mouse anti-C5a-biotin (1:250, Biozol, Eching, Germany, #BLD-518306). Peroxdiase-conjugated anti-species antibodies were used for detection (1 h, PBS-T): goat anti-mouse (1:5000, Jackson ImmunoResearch, West Grove, PA, USA, #115-035-164), rabbit anti-goat (1:5000, Jackson ImmunoResearch, #305-035-003). Visualization was performed by WesternSure PREMIUM Chemiluminescent Substrate (LI-COR, Bad Homburg, Germany, #926-95000) in a Fluor Chem FC2 Imaging System (Alpha Innotech, San Leandro, CA, USA).