Ecl chemiluminescence reagent

To extract protein, cells were washed in phosphate-buffered saline (PBS) then lysed in RIPA buffer (Sigma) supplemented with Complete protease inhibitors (Roche), before centrifugation to remove cell debris. Protein concentrations were then quantified using Bradford assay reagent (BioRad), and 40ug of protein per sample was run on a 4–12% Tris Glycine polyacrylamide gel (Invitrogen). The gel was then transferred to a PVDF membrane. The membranes were then incubated in Tris-buffered saline with 0.1% Tween 20 (TBST) with a primary detection antibody for 2-hrs, before washing with TBST. Primary antibodies used were anti-CTCF (clone D31H2, Cell Signaling) and beta-actin (ab49900, Abcam). Primary antibody staining was followed by incubation with an HRP-linked anti-rabbit antibody. Bands were the imaged by incubation of the membrane with ECL chemiluminescence reagents (ThermoFisher) and imaging on a ChemiDoc MP imager (BioRad). For flow cytometry for murine Heat Shock Antigen (HSA), cells were washed in PBS before staining with anti-HSA-APC (Biolegend) at 1:200 dilution for 30mins at 4C, before washing in PBS and analysis using a Fortessa flow cytometer (Becton Dickson).

Mouse brains were homogenized in liquid nitrogen and protein was extracted using RIPA Lysis and Extraction Buffer containing three protease inhibitors (Thermo Scientific, Waltham, MA, United States). Western blot analyses were performed as previously described (Qiu et al., 2020 (link)). Antibodies for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cat# 2118,1:1,000), MyD88 (cat# 4283,1:1,000), p-P38 (cat# 9211,1:1,000), P38 (cat# 8690, 1:1,000) and p-Akt (cat# 4060, 1:500), were purchased from Cell Signaling Technology (Danvers, MA, United States), p-JNK (cat# sc-12882, 1:500), JNK (cat# sc-7345, 1:500), p-ERK (cat# sc-7383,1:500), ERK (cat# sc-514,302, 1:500) and Akt (cat# sc-81434, 1:500) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, United States) and antibodies for beta-amyloid (clone 6E10, 1:1,000) were purchased from BioLegend (San Diego, CA, United States). Antibodies for AT8 (cat MN1020, 1:1000) were purchased from Thermo Scientific (Waltham, MA, United States). Blots were visualized using ECL chemiluminescence reagents from Thermo Fisher. All bands were quantitatively analyzed using Image J (ImageJ, RRID: SCR_003070).

Mouse hippocampi were homogenized in chilled TBS supplemented with a protease- and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentrations of the homogenates were determined by BCA assay (CWBIO). Samples were electrophoretically separated on 10 or 12% SDS–PAGE gels and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% nonfat milk in TBS containing 0.1% Tween-20 (TBS-T) for 1 h and probed with primary antibodies (Anti-UCP-1 antibody ab10983) at 4 °C overnight. The membranes were subsequently developed with the corresponding horseradish peroxidase-conjugated secondary antibody and ECL kit (Thermo Fisher Scientific). Densitometry quantification of the protein bands was analyzed using IMAGE-PRO PLUS 6.0 imaging software.
The mixture of proteins was extracted from scapular brown adipose tissues in RIPA buffer (0.5% NP40, 0.1% sodium doxycycline, 150 mM NaCl, 50 mM Tris.HCl [pH 7.4]) containing a complete protease inhibitor cocktail (Abcam). PVDF membranes were probed simultaneously with primary antibodies of UCP1 (Abcam) and β-tubulin (Abcam). The protein bands were visualized with ECL chemiluminescence reagents (Thermo Fisher Scientific) and quantified using IMAGE-PRO PLUS 6.0 imaging software.

The protein regulation of HSP90AA1, CDK2, STAT3, and p-STAT3 by YQYJ was estimated by Western blotting. RIPA buffer was used to extricate the total protein of experimental groups. GAPDH was used as the internal standard. Finally, the protein bands were detected using ECL chemiluminescence reagents and biomolecular imagers (Thermo Fisher Scientific, USA). The details of the experiment are shown in Supplementary Information S1.

To identify the ΔNcGRA6 strain at the protein level, we extracted the protein from the ΔNcGRA6 strain and Nc-1 strain to detect NcGRA6 and NcSRS22A protein levels via western blot analysis. A rat polyclonal antibody against NcGRA6 and a mouse monoclonal antibody against the putative surface antigen NcSRS22A were prepared and constructed in our laboratory. First, the purified ΔNcGRA6 strain and Nc-1 strain tachyzoites were lysed using RIPA buffer (Beyotime, China) supplemented with 1 mM PMSF protease inhibitor. Then, the lysates were mixed with 5 × SDS-PAGE Sample Loading Buffer (Beyotime, China), boiled for 5 min, and electrophoresed on a 12% (w/v) SDS-PAGE gel. After electrophoresis, the protein bands were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA) using a Mini Trans-Blot® 25Electrophoretic Transfer Cell (BIO-RAD, USA). The membranes were blocked with 5% (w/v) skim milk at 4°C overnight, incubated with primary antibody targeting NcGRA6 or NcSRS22A (1:200) for 2 h at 37°C, followed by incubation with HRP-conjugated secondary goat anti-rat IgG (H+L) or goat anti-mouse IgG (H+L) (Earthox, USA, 1:5,000) for 1 h at 37°C, respectively. Finally, the bands were detected using ECL chemiluminescence reagents (Thermo, USA) and visualized with an ECL western blot Detection System (Clinx Science Instruments Co., Ltd., China).