Ab97051

Total protein content was extracted from SH-SY5Y cells or EVs using RIPA kits (R0010, Solarbio, Beijing, China). Briefly, 40 µg of each sample was extracted, separated with 10% SDS-PAGE, and electrotransferred onto a PVDF membrane (Merck Millipore, Billerica, MA, USA). Subsequently, the membrane was blocked and incubated with the following primary antibodies: EGR1 (dilution ratio of 1:1000, #4154, CST, Beverly, MA, USA), NOX4 (dilution ratio of 1:2000, ab109225, Abcam), p–p38 (dilution ratio of 1:1000, #4511, CST), P38 (dilution ratio of 1:1000, #8690, CST), TH (dilution ratio of 1:5000, ab137869, Abcam), 4-HNE (dilution ratio of 1:1000, ab46545, Abcam), and GAPDH (dilution ratio of 1:10000, ab128915, Abcam). Afterward, the membrane was incubated with IgG (dilution ratio of 1:10000, ab97051, Abcam). Later, the membrane was developed using an electrogenerated ECL, and then the membrane was exposed to light using the Image Quant LAS 4000 C gel imager (GE Company, Schenectady, NY, USA) and analyzed with the ImageJ software (1.48 u, National Institutes of Health, Maryland).

After transfection for 48 h, proteins were extracted from cultured cells using RIPA buffer (Thermo Fisher Scientific) containing protease inhibitor. Then, the protein concentration was assayed by BCA protein assay kit (Thermo Fisher Scientific). The high-temperature denatured protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V and transferred onto the polyvinylidene fluoride (PVDF) membranes (Amersham, USA). The membranes were incubated with primary antibodies overnight at 4°C after being blocked for 1 h. Then, the membranes were incubated with horseradish peroxidase labeled secondary antibody goat anti-rabbit IgG H&L (ab97051, 1:2000, Abcam, Cambridge, UK) at room temperature for 1 h. Afterwards, the membranes were washed with TBST buffer for three times. The primary antibodies included rabbit polyclonal anti-E2F7 (ab56022, 1:1000, Abcam) and rabbit polyclonal anti-GAPDH (ab9484, 1:1000, Abcam). All proteins were visualized using an optical luminometer (GE, USA). The relative expression of proteins was analyzed by using the Image Pro Plus 6.0 (Media Cybernetics, USA). All experiments were conducted in triplicate.

Total protein was isolated from tissues or cells (48 h after transfection) using radioimmunoprecipitation assay buffer. The protein was separated by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Afterward, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, and then blocked with 5% skim milk powder at room temperature for 1 h. Subsequently, the PVDF membrane was incubated with the corresponding diluted antibody overnight at 4°C. The following primary antibodies were used: rabbit antibodies to STC1 (1:1000, ab83065, Abcam, Cambridge, United Kingdom); p-JNK (1:1000, ab124956, Abcam); JNK (1:2000, ab208035, Abcam); bone morphogenetic protein-2 (BMP2; 1:1000, ab14933, Abcam). Osteocalcin (1:500, ab93876, Abcam). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ab9485, 1:2500) was used as a normalization medium. The blot was incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody (ab97051, 1:2000, Abcam) for 1 h. Protein bands were visualized using the enhanced chemiluminescence detection kit (No. BB-3501, Amersham Pharmacia Biotech, Chicago, IL, United States) and Bio-Rad Image analysis system (Bio-Rad Laboratories, Inc., Hercules, CA, United States). Software Quantity One v4.6.2 was used for the further quantifying analysis.

Total protein colon samples were extracted using RIPA buffer (P0013C, Beyotime, Shanghai, China), and 15 μg proteins were separated by 10% SDS-PAGE electrophoresis and transferred to a methanol-activated PVDF membrane (GVPPEAC12, Millipore, Bellerica, USA). The membrane was blocked for 1 h in PBST containing 5% milk and subsequently probed with Occludin antibody (GTX114949, GeneTex, Texas, USA), ZO-1 antibody (Ab96587, Cambridge, UK), P65 antibody (YT3108, ImmunoWay, Texas, USA), phosphorylated P65 antibody (B4568, ImmunoWay, Texas, USA), and GAPDH antibody (ab9485, Abcam, Cambridge, UK), respectively, for 2 h. After 1 h incubation with goat-anti-rabbit HRP-conjugated secondary antibody (ab97051, Abcam, Cambridge, UK), the protein bands were detected with luminal reagent (Millipore, Pittsburgh, USA) and their relative intensities were quantified using Image J. Biological verifications were performed three times in each group.

Samples were separated in 10% polyacrylamide gels at 150 V for 1 h, before being transferred to a PVDF membrane at 110 V for 1 h. The membrane was then blocked with 5% non-fat milk in TBST buffer for 1 h, incubated overnight with 1:1000 rabbit polyclonal anti-pneumolysin primary antibody (ab71811, abcam, Cambridge, United Kingdom) at 4 °C with agitation, followed by 1 hr incubation with 1:5000 goat polyclonal anti-rabbit HRP-conjugated secondary antibody (ab97051, abcam, Cambridge, United Kingdom) at room temperature with agitation, complete with TBST-washing cycles prior and after. Blots were incubated with Bio-Rad Clarity^TM Western ECL Substrates and the resulting bands were visualized in a Bio-Rad ChemiDoc^TM Touch imaging system in Chemiluminescence mode (Bio-Rad, Hercules, California, United States). The experiment was performed in triplicate. Technical repeats were taken to ensure consistent results were obtained.

Total protein was extracted by sonication of cerebral cortex tissue for 10 s at 4 °C, 50% power capacity (Heat System Ultasonic INC) in 150 μl lysis buffer: 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1% (V/V) β-mercaptoethanol, 1 mM PMSF, 1 μl protease inhibitors cocktail and 1 μl phosphatase inhibitors cocktail (all chemicals from Sigma-Aldrich, St. Louis, MO). After centrifugation at 10,000×g for 15 min at 4 °C the supernatant was collected and protein concentration determined spectrophotometrically using NanoDrop 2000 (Thermo Scientific, Waltham, MA). Western-blotting was performed according to a standard protocol used in our lab^{40 (link)} on 10% polyacryl-amid gel and transferred to PVDF membrane. Each sample was tested in duplicates of 10 and 20 µg/lane, to verify linearity. Primary antibodies and their dilutions in TBST (Tris-buffered saline supplemented with Tween-20) were: p62, ab91526–1:1500, Abcam, Cambridge Science Park, Cambridge, UK; Beclin-1, #3495–1:1000, Cell Signaling Technology, Danvers, MA; LC3, L7543–1:1000, Sigma-Aldrich, ibid. Secondary goat-anti rabbit antibody ab97051–1:10000, Abcam, ibid, diluted in TBST.