Anti β actin

Protein extracts (25 µg) prepared with Pierce™ IP Lysis buffer were resolved by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Pall Corporation, FL, USA). Membranes were blocked with PBS containing 0.02% Tween 20 and 5% nonfat dry milk and blotted overnight with primary antibodies against proteins of interest (anti-FAK, anti-p-FAK-tyr-925, anti-p-FAK-tyr397, anti-AKT, anti-p-AKT, anti-ERK, anti-p-ERK, anti-JNK, anti-p-JNK (Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin from Abfrontier (San Diego, USA). HRP-conjugated goat anti-rabbit IgG purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) was used as the secondary antisera at a 1:5000 dilution. Protein bands were visualized using a chemiluminescence imaging system, Ez-Capture MG (ATTO, NY, USA).
Reciprocal IP was performed using HCT-116 lysates (1500 µg total protein) and antibodies against zyxin, nesprin-1, and desmoplakin (Abcam, Cambridge, UK). Immunoprecipitated proteins were resolved by SDS-PAGE, and FAK was detected by Western blotting.

MCF-7 cells were cultured overnight at a density of 3 × 10⁵ cells/well. The cells were then treated either with a CHE or with vehicle. Next the samples were homogenized using 400 μL lysing buffer containing 150 mmol/L KCl, 10 mM Tris, pH 7.4, 1% Triton X-100, and protease inhibitor cocktail (Complete Mini, Roche, Mannheim, Germany). The protein concentration of each cell homogenates was determined as described previously [21 (link)]. Samples consisting of 30–50 μg of protein were separated on 10% SDS-polyacrylamide gels by electrophoresis and thereafter transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% bovine serum albumin and probed with specific primary antibodies, namely, anti-ERα (Stressgen Biotechnologies Inc., Victoria, BC, Canada), anti-pHER2/anti-tHER2 (IPVH00010, Millipore, Bedford, MA, USA), anti-α-tubulin (AbFrontier, Seoul, Korea), and anti-β-actin (AbFrontier, Seoul, Korea). The immunoreactive bands were visualized using enhanced chemiluminescence detection reagents (Thermo Scientific, Bremen, Germany) and quantified by Multigauge software analysis (Fuji Photo Film Co., Ltd., Tokyo, Japan).

Briefly, 20 fly heads for each genotype were homogenized in RIPA buffer, and lysates were loaded in each lane of 10% SDS gels and transferred to nitrocellulose membrane. Membranes were blocked in 5% BSA and incubated with primary antibodies at 4 °C overnight. After washing membranes with TBS-T, membranes were incubated with the appropriate secondary antibody. Using the ECL Western blotting detection reagent, membranes were developed and images were captured using FluorChem E image processor. Antibodies used were anti-Tau (1:1000, T46, Cat no. 13-6400, Invitrogen), anti-AT180 (1:1000, Cat no. MN1040, Invitrogen), anti-PHF-1 (1:1000, Cat no. MN1050, Invitrogen), anti-AT8 (1:1000, Cat no. MN1020, Invitrogen) and anti-β-actin (1:1000, Cat no. JLA20, DHSB). β-actin was used as a loading control. Signal intensity was quantified using ImageJ (NIH) software. Flies used were 30 days old after eclosion. For Western blot analysis of SH-SY5y cells, the following antibodies were used: anti-Tau (Cat no. ab64193, Abcam), anti-β-actin (Cat no. LF-PA0207, AB Frontier), anti-Myc (Cat no. C3956, Sigma), anti-HA (Cat no. H6908, Sigma), and anti-CHIP (Cat no. sc-133066, Santa Cruz Biotechnology).

Mouse tissues were lysed with using RIPA buffer (Elpis-Biotech, Daejeon, Korea) and subjected to immunoblot analysis as described previously [46 (link)]. Proteins from liver whole and cytosolic fraction lysates were separated by 10% SDS- PAGE and transferred to nitrocellulose membranes. The membranes were probed with anti-β-actin (AbFrontier; diluted 1:5000) [26 (link)], anti-CYP2E1 (Proteintech, 19937-1-AP, 1:1000), anti-Cleaved caspase3 (Cell signaling, #9661; diluted 1:1000), anti-Cytochrome C (Cell signaling, #11940; diluted 1:1000), anti-smac (Abcam, ab32023; diluted 1:1000), and anti-α-tublin (AbFrontier; diluted 1:5000). The membranes were probed with specified antibody. Immunoreactive proteins were visualized using an Amersham ECL kit (GE Healthcare, Piscataway, NJ, USA) or using iBright CL1000 imaging system (Invitrogen) according to the manufacturer's instructions.

The samples (cells and brain tissues) were mixed with radioimmunoprecipitation assay (RIPA) buffer (Sigma), 1 × proteinase inhibitor cocktail (Sigma), and 5 × loading buffer, incubated at 100 °C for 10 min, and centrifuged at 14,000×g for 10 min to remove the debris. The prepared samples were separated with SDS-PAGE and blotted onto membranes. The blotted membranes were incubated with anti-APP C-terminal (Sigma, A8717) or anti-β-actin (AbFrontier, Seoul, Korea, LF-PA0207) at 4 °C overnight. They were then incubated with a conjugating secondary antibody. Protein bands were visualized using an ECL kit (Dogen, Seoul, Korea).