α gapdh

Cells were lysed in RIPA buffer (150 mM NaCl, 10 mM Tris, pH7.4, 0.1% SDS, 1% Triton X-100, 5 mM EDTA, 1% Na-deoxycholate, 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL pepstatin). The primary antibodies used were α-Flag (Sigma-Aldrich, F4042, 1:2000 or Sigma-Aldrich, F7425, 1:1000), α-HA (BioLegend, #901501, 1:1000), α-p38 (Cell signaling, #9212, 1:1000), α-pp38 (Cell signaling, #9219, 1:1000), α-MKK3 (Cell signaling, #5674, 1:500), α-MKK6 (Abnova, #M02, 1:500), α-MAPKAPK2 (Cell signaling, #12155, 1:1000), α-PRMT1 (Sigma-Aldrich, #P16220, 1:1000), α-GAPDH (Cell signaling, G9545, 1:10000), α-mono- and di-methyl arginine (abcam, ab412, 1:500). The secondary antibodies used were anti-mouse (Sigma-Aldrich) or anti-rabbit (Genetex) IgG linked-horseradish peroxidase.

The following antibodies were used: α-Myc (9E10 Santa Cruz #sc-40), α-Flag (M2 Sigma #F3165), α-Nct (Cell Signaling 3632S), α-GAPDH (Cell Signaling Technology, ab125247), α-APP (C7), α-AICD 50–99 (Rb) was a kind gift from Philip Szekeres at Eli Lilly, α-Ms 800nm (Licor Bio 926–32212) and α-Rb 680nm (Licor Bio 926–68021). Tripeptides were synthesized by Anaspec corp. Total brain lipid extract was from Avanti Polar Lipids (#131101). The following Aβ ELISA kits were used: 4G8 (Meso Scale Diagnostics, K15199E) or 6E10 (Meso Scale Diagnostics, K15200E) for cell-based assays; Aβ40 (#KHB3482) and Aβ42 (#KHB2442) ELISA kits from Invitrogen for in vitro assays.

The supernatants of the treated cells were precipitated by the chloroform–methanol method, as previously described [34 ]. Pellets were obtained by lysing cells in 25 mM of Tris-HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, and 1% sodium dodecyl sulfate (SDS), plus protease and phosphatase inhibitors. The protein concentration was determined by the BCA protein assay. Samples were separated in a 14% SDS-polyacrylamide gel electrophoresis gel, transferred to a nitrocellulose membrane, and blocked with 5% skimmed milk for 1 h. Then the membrane was incubated overnight with primary antibodies: α-casp1 (1:1000; Cell Signaling 2225), α-IL-1β (1:1000; Acris R1130P), α-NLRP1 (1:1000; ALX-210-904-R100), α-NLRP3 (1:1000; Cell Signaling 15101), α-ASC (1:1000; Santa Cruz Biotechnology sc-22514-R), α-gapdh (1:1000; Cell Signaling 2118), and α-tubulin (1:3000; Abcam ab6160) at 4 °C. Membranes were washed and probed with the appropriate secondary antibody conjugated with peroxidase for enhanced chemiluminescence detection (Amersham Pharmacia Biotech).

Primary antibodies used in this study include: α-GAPDH (1:2500, 14C10, Cell Signaling Technology, Danvers, MA), α-Progesterone Receptor A/B (1:1000, 8757, Cell Signaling Technology), α-HER2 (1:1000, 2165, Cell Signaling Technology), α-Estrogen Receptor α (1:1000, 8644, Cell Signaling Technology), α-Vimentin (1:1000, MA5-11,883, ThermoFischer Scientific), α-Ecadherin (1:1000, ECCD-2, #13–1900, ThermoFischer Scientific) and α-Ki67 (1:1000, #9449 Cell Signaling Technology).

The mouse mAb S16 was produced by Sugawara et al.^{27 (link)}. BALB/c mice were immunized with 50 μg purified HEF proteins of C/Ann Arbor/1/50 in Freund’s complete adjuvant thrice at weekly intervals. The hybridoma cells secreting antibodies to HEF were screened using enzyme-linked immunosorbent assay (ELISA). The isotype of S16 was classified as IgG2a using double immunodiffusion.
For western blotting, α-mouse ACAA2 (ab128911; Abcam, Cambridge, UK), α-GAPDH (#5174; Cell Signaling Technology, Danvers, MA, USA), α-HSP60 (#12165; Cell Signaling Technology), α-histone H3 (#4499; Cell Signaling Technology), α-Flag M2 (F1804; Sigma-Aldrich, St. Louis, MO, USA), and α-mouse albumin (#4929; Cell Signaling Technology) polyclonal antibodies (pAbs) were used as the primary antibodies. Horseradish peroxidase (HRP)-conjugated secondary antibodies against mouse and rabbit IgG were purchased from Bio-Rad (Hercules, CA, USA). The pAb against mouse ACAA2 (TA321746; OriGene, Rockville, MD, USA) and HRP-conjugated goat α-mouse IgG2a antibody (Southern Biotech, Birmingham, AL, USA) were used for ELISA. The IgG2a isotype control (BioLegend, San Diego, CA, USA) was used as a control antibody for the indirect immunofluorescence assay and injection of S16 into mice.