Rabbit anti pdi

Brains were dissected immediately following euthanization. One hemisphere was then homogenized in protein lysis buffer (PLB: 50 mM Tris pH 6.8, 150 mM NaCl, 20 mM EDTA, 1 mM EGTA, 0.5% SDS, 0.5% NP40, sarkosyl 0.5%, 1 mM prefabloc, 10 mM orthovanadate, 2.5 mM sodium fluoride) [37] (link), [38] (link). Total protein concentrations were then determined using the bicinchoninic acid assay (Thermo Scientific, Rockford, IL). Brain homogenates and lysates were both stored in aliquots at −20°C.
Freshly thawed brain homogenates and cell lysates were each diluted to 15 µg/µL and loaded onto either 8.5 or 10% SDS PAGE gels, transferred to 0.45 µm PVDF membranes (Immobilon-P, Millipore, Billeria, MA) and probed with the following primary antibodies: mouse anti-FLAG (#F3165 Sigma Aldrich, St. Louis, MO), mouse anti-ubiquilin (clone 3D5E2, Invitrogen, Carlsbad, CA) which recognizes ubiquilin proteins, rat anti-BiP (sc-13539 Santa Cruz Biotechnology, Santa Cruz, CA), and goat anti-actin (Santa Cruz Biotechnology), rabbit anti-PDI (#2446 Cell Signaling, Danvers, MA), mouse anti-CHOP (#2895 Cell Signaling). Secondary antibodies used were horse radish peroxidase conjugated goat anti-mouse (#31430), goat anti-rabbit (#31460), goat anti-rat (NA935V GE Healthcare) and rabbit anti-goat (#31492 Thermo Scientific).

Sulfo-NHS-LC-biotin, streptavidin agarose resin beads, Dynabeads Protein G, Turbofect Transfection Reagent and restriction enzymes were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). Glutathione Sepharose 4B was purchased from GE Healthcare Life Sciences (Piscataway, PA, USA). The γ-secretase inhibitor Compound E was purchased from Millipore (San Diego, CA, USA). 6-carboxyfluorescein (FAM)-Aβ was purchased from AnaSpec (Fremont, CA, USA). The following antibodies were used in this study: mouse anti-Myc (for immunoblot, Life Technologies, Carlsbad, CA, USA); rabbit anti-Myc (for immunostaining) and rabbit anti-PDI (Cell Signaling Technology, Danvers, MA, USA); sheep anti-Myc (for immunostaining, Thermo Fisher, Carlsbad, CA, USA); sheep anti-TGN46 (GeneTex, Irvine, CA, USA); mouse anti-nicastrin (Abcam, Cambridge, MA, USA); rabbit anti-PS1-NTF (Ab14) antibody (developed in-house previously); mouse anti-PS1 loop (Millipore); goat anti-human TREM2 and sheep anti-mouse TREM2 (R&D Systems, Minneapolis, MN, USA); mouse anti-β-actin (Sigma, St Louis, MO, USA); rabbit anti-GST, normal mouse IgG and rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA).

Digitonin was obtained from Merck (Darmstadt, Germany), lactacystin was purchased from Enzo Life Sciences (Farmingdale, NY, USA), and epoxomicin was purchased from UBPBio (Oxfordshire, UK). The mouse monoclonal anti-HA antibodies, anti-rabbit Alexa594 and anti-mouse Alexa488 were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The mouse monoclonal anti-EDEM1, monoclonal anti-β-actin-peroxidase, rabbit anti-APP C-terminal, as well as the secondary anti-rabbit HRP and anti-mouse HRP antibodies were from Merck (Darmstadt, Germany). The mouse monoclonal anti-APP were obtained from OriGene (Rockville, MD, USA), the rabbit anti-PDI were from Cell Signaling (Danvers, MA, USA). The mouse anti-calreticulin was from Enzo Life Sciences (Farmingdale, NY, USA), whereas mouse monoclonal anti-calnexin was from BD Biosciences (Bedford, MA, USA). The mouse anti-HSP70 was obtained from Santa Cruz Biotechnology (Dallas, TX, USA).