Chemidoc detection system

After 24 h of treatment with acetylshikonin (0, 1.25, 2.5, and 5 μM), total protein from A498 and ACHN cells was extracted using RIPA buffer (Sigma, St. Louis, MO, USA) with protease and phosphatase inhibitors, PMSF phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO, USA). Proteins were loaded on SDS-PAGE gels with appropriate percentage according to protein size and blotted onto polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 3% bovine serum albumin (BSA; Bovogen, Victoria, Australia) for 30 min at room temperature (25°C), and immunoblotting was performed with specific primary antibodies at 4°C overnight. Membranes were then incubated with HRP-tagged secondary antibodies for 1 h at room temperature (25°C). Protein bands were visualized by using the enhanced chemiluminescence (ECL; Gendepot, Barker, USA) and detected with Chemi-doc detection system (Bio-Rad, Hercules, CA, USA).

Simultaneous detection of multiple cytokines was obtained using the Mouse Inflammation Antibody Array (Raybiotech, Norcross, GA; Canada; AAM‐CYT‐6). Primary microglia from B6129 mice were plated in a 6‐well at the concentration of 6.5 × 10⁵ cells/well in culture medium. After 18 hr, cells were serum starved for 4 hr, and later treated with Aβ 1 µM or 100 ng/ml NGF or Aβ and NGF simultaneously. Cells were lysed in ice‐cold RIPA buffer (50 mM Tris/HCl, 150 mM NaCl, 1 mM EDTA, 1% Igepal, 0.5% Sodium Deoxycholate, 0.1% SDS, Protease Cocktail inhibitor) and sonicated briefly, and then collected by centrifugation at 13,000 rpm at 4°C for 15 min. Arrays were incubated with the appropriate blocking buffer for 2 hr. Eighty µg of protein extract were diluted in blocking buffer and incubated with the array overnight at 4°C. Then, arrays were washed accordingly and incubated for 3 hr at room temperature with the Biotinylated Antibody Cocktail solution. After washing, arrays were incubated with HRP‐streptavidin for 2 hr and detected using the Detection Buffer. Images were captured using the Chemidoc detection system (Bio‐Rad).

The other hemisphere of the brain was snap‐frozen, homogenized, and lysed in buffer containing protease and phosphatase inhibitors (ThermoFisher) and analyzed by Western blotting, as described.⁶¹, ⁶² In brief, the lysates were centrifuged at 14,000 rpm for 25 min at 4°C. Supernatant protein concentrations were then determined using the BCA method (Cat. #23227, Thermo Scientific), and the samples were then boiled for 5 min in Laemmli's sample buffer (Cat. # 1610737, Bio‐Rad). Equal amounts of protein were separated on SDS‐polyacrylamide gels (Cat. Cat No: #4561105, Bio‐Rad) and then transferred to polyvinylidene fluoride membranes (Cat. No: IPVH00010, Immobilon‐P, Thermo). The membranes were then blocked with TBST solution (50 mM Tris‐HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 5% non‐fat dry milk at 4°C for 1 h. The membranes were then incubated overnight at 4°C with anti‐NeuN (1:1000; mouse monoclonal, Cat. #104224, Abcam) and anti‐GAPDH (1:1000; Cell Signaling, 2118S) in 3% BSA. After washing, the membranes were incubated with secondary antibody diluted in 3% BSA. Blots were imaged using a ChemiDoc detection system (BioRad), and signals were quantified using Image Lab software (BioRad).

0.02 g of carotid atheroma plaque was washed with PBS and cut at 300 µm with McIllwain Tissue Chopper (The Mickle Laboratory Engineering Co. LTD.) and the resulting mixture was diluted in 100 µl RIPA buffer (150 mM NaCl; 50 mM Tris-Cl, pH 7.5; 1% NP-40; 0.5% deoxycholate; 0.1% sodium dodecyl sulphate) containing protease inhibitors. Samples were homogenized for 1 h and 30 min on a rotator at 4°C followed by centrifugation for 15 min at 14800 rpm. The supernatants were collected and 10 µl of sample was subjected to 15% SDS-PAGE. Proteins were electrophoretically transferred to a PVDF membrane and blocked overnight. Then, membranes were incubated with rabbit anti-LC3B (D11) antibody (Cell Signalling) or mouse anti-GAPDH (6C5) (Millipore) followed by incubation with anti-rabbit or anti-mouse horseradish peroxidase (HRP) conjugate secondary antibody. Bound antibodies were detected with SuperSignal substrate (Thermo Scientific) on a Chemidoc detection system (BioRad). Signals were quantified by densitometric scanning with the Chemidoc software and densitometric values were normalized against GAPDH. Statistical significance was determined by using the non parametric Mann-Whitney U test.

Organs were collected from adult male mice and homogenized using beads in Tissue Lyzer II (Qiagen, Germany) in NETN400 Lysis buffer (0.5% NP-40, 50 mM Tris–HCl pH 8.0, 2 mM EDTA, 400 mM NaCl, 10 mM NaF, 50 mM β-glycerophosphate) containing protease inhibitors. Protein lysates were centrifuged at 4 °C at 3000 rpm for 5 min. The supernatant was carefully removed and pellets containing large plasma membrane pieces, DNA and nucleoli were diluted with NETN0 Lysis buffer (no containing salts) to 100 mM NaCl. Protein concentration of nuclear fractions were determined by Pierce BCA Protein Assay Kit (Thermo Scientific, USA). Samples were loaded on 8% SDS-PAGE gel and transferred onto nitrocellulose membrane (GE Healthcare Life Science, Germany). Membranes were blocked for at least 1 h in 5% milk in TBS-T before incubating overnight at 4 °C with the appropriate primary antibody. Antibodies used were anti-ZNF644 and anti-GAPDH (G9545, Sigma-Aldrich, USA). The anti-ZNF644 (raised against N-terminus AA50-602) was kindly provided by Xiaochun Yu. The following day, membranes were washed with TBS-T, incubated with appropriate secondary antibody (Sigma-Aldrich, USA) at room temperature for 1 h and then washed again with TBS-T. Membranes were developed using Pierce™ ECL Western Blotting substrate (Thermo Scientific™, USA) and images captured using a ChemiDoc™ detection system (Bio-Rad).

Tumor and normal tissues were lysed in RIPA buffer (Solarbio, Beijing, China), after which phosphatase and protease inhibitors were added and denatured for 15 minutes at 100°C. The protein samples were extracted with SDS-PAGE at a concentration of 10%, and the separated proteins were transferred into polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with a solution of skim milk powder at a concentration of 5% for 1 hour and incubated with primary antibodies, including anti-NECAP2 antibody (1 : 200, Proteintech, 15899-1-AP), anti-GAPDH antibody (1 : 5000, Abcam, ab9485) overnight. After thorough washing, the membranes were incubated with secondary antibodies (1 : 2000, Proteintech, SA00001-2) for 2 hours at ambient temperature and observed with the ECL kit chemiluminescence reagent (Billerica Millipore, MA, USA). The Chemidoc detection system (Bio-Rad, Hercules, California, USA) was employed to identify protein band signals. The ImageJ program (National Institutes of Health, USA) was used to quantify these signals.

Protein was extracted using RIPA buffer (Sigma) with a protease inhibitor cocktail (cOmplete Mini EDTA-free, Roche) and a phosphatase inhibitor cocktail (PhosSTOP EASYpack, Roche) [21 (link)]. We used the following primary antibodies: anti–NFKB-p65 (1:2000; Abcam #ab16502), anti–NFKB-p65 (phospho-Ser536) [93H1] (1:1000; Cell Signaling Technology #3033), anti-IKKa [Y463] (1:5000; Abcam #ab32041), anti-IKKa (phospho-Ser32/36) [5A5] (1:1000; Cell Signaling Technology #9246), anti-p44/4S2 MAPK [137F5] (1:2000; Cell Signaling Technology #4695), anti-p44/42 MAPK (phospho-Thr202/Tyr204) [E10] (1:2000; Cell Signaling Technology #9106), anti-LC3B (1 mg/mL; Abcam #ab48394), EGFR (#4267, CST), pEGFR (#2234, CST) and β-actin (#A5316, Sigma). β-actin protein expression was used as a loading control. Horseradish peroxidase-conjugated secondary antibodies were used for chemiluminescence-based detection of protein expression in the ChemiDoc detection system (BioRad).