Anti rabbit igg hrp

The following primary antibodies were used: custom rabbit polyclonal anti-AQP4 (GenScript Biotech, Piscataway, NJ, USA) at a concentration of 0.4 μg/mL for immunoblot analysis and 0.7 μg/mL for immunofluorescence and custom rabbit polyclonal anti-mouse AQP4ex generated against the peptide DSTEGRRDSLDLASC within the mouse AQP4 carboxy-extension (GenScript Biotech, Piscataway, NJ, USA) at a concentration of 0.5 μg/mL for immunoblotting and immunofluorescence [16 (link)]. For immunofluorescence, AlexaFluor 488 anti-rabbit was used (Life Technologies, Thermo Fisher Scientific, Carlsbad, CA, USA) at a concentration of 1 μg/mL; for immunoblotting, anti-rabbit IgG-HRP (Bio-Rad, Hercules, CA, USA) was used following the manufacturer’s instructions.

H3K27ac (39,133, Active Motif Lot # 31814008), H3K27ac (39,685, Active Motif, no. 14517014), H3K18ac (ab1191, Abcam, no. GR3211480–1), H3K27me3 (9756, Cell Signaling), H3K9ac (ab4441, Abcam), H4K16ac (39,167, Active Motif), H3 general (ab1791, Abcam, no. GR177884–2), Spike-In antibody (61,686, Active Motif, Lot# 00419007), p300 (sc-584, Santa Cruz, Lot # F3016), Gapdh (2118, Cell Signaling, Lot # 10), CBP(D6C5) (7389S, Cell Signaling), p53(CM5) (NCL-L-p53-CM5p, Leica Biosystems), PKCs p2056 (ab18192, Abcam), KAP1/TRIM29 pS824 (A300-767A, Bethyl), Acetyl-p53 (Lys379) (2570, Cell Signaling), anti-mouse IgG-HRP (NA93V, GE, no.9773218), anti-rabbit IgG-HRP (170–6515, Bio-Rad, no. 350003248).

Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature and then incubated with primary antibodies diluted in blocking buffer on a shaker for 1 to 2 h at room temperature or overnight at 4 °C. The membranes were incubated with horseradish peroxidase-labeled secondary antibody for 1 h. Ultrasensitive enhanced chemiluminescence (ECL) substrate was used for protein detection. Primary antibodies used were anti-FMNL2 (1:250, mouse monoclonal, SCB, Heidelberg, Germany) and anti-Tubulin (1:1000, rabbit monoclonal, CST, Leiden, The Netherlands). Secondary antibodies used were anti-rabbit IgG-HRP (1:5000, goat, Biorad, Feldenkirchen, Germany) and anti–mouse IgG-HRP (1:5000, goat, GE Healthcare, Chicago, IL, USA).

The following primary antibodies were used: rabbit polyclonal anti-AQP4 (Sigma, Saint Louis, Missouri, USA) diluted to 1:2000 for immunoblot analysis and 1:1000 for immunofluorescence; rabbit polyclonal anti-α1-Syntrophin (Sigma, Saint Louis, Missouri, USA) at 1:1000 for immunofluorescence experiments; mouse monoclonal anti-human Dystrophin (Monosan, PB Uden, The Netherlands) used at 1:500 for immunofluorescence; mouse monoclonal anti-CD31 (Dako, Santa Clara, California, USA) diluited to 1:100 for immunofluorescence experiments; Custom rabbit polycolonal anti mouse AQP4ex generated against the peptide DSTEGRRDSLDLASC within the mouse AQP4 carboxy-extension (GenScript) and diluted 1:5000 for immunoblotting and 1:4000 for immunofluorescence. For immunofluorescence, AlexaFluor 488 anti-rabbit, AlexaFluor 488 anti-human, AlexaFluor 488 anti-mouse and AlexaFluor 594 anti-mouse were used (all from Life Technologies, Thermo Fisher Scientific, Carlsbad, California, USA) at a dilution of 1:1000; for immunoblotting, anti-rabbit IgG-HRP (Bio-Rad, California, USA) was used at 1:3000.

Proteins were extracted from the exosomes, liver tissue, and AML12 cells using a RIPA lysis buffer (Rockland Immunochemicals Inc., PA, United States) that included protease and phosphatase inhibitors (Thermo Fisher Scientific). First, the proteins were quantified using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, United States), then 50–100 μg of protein were separated using 10% Mini-PROTEAN^® TGX™ Precast Protein Gel (Bio-Rad Laboratories, Hercules, CA, United States) and electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA, United States). The membranes were blocked using EveryBlot blocking buffer (Bio-Rad Laboratories, Hercules, CA, United States) for 5 min at room temperature (RT) and were then incubated with primary antibodies against CD63, IRE1a (Invitrogen), p-IRE1a (LSBio, Seattle, WA, United States), PI3K p85, p-PI3K, Akt, p-Akt, β-actin, acetyl-CoA carboxylase (ACC), p-ACC, fatty acid synthesis (FAS), and XBP1s (Cell Signaling, MA, United States) for 12 h at 4°C. Subsequently, the membranes were incubated with a secondary antibody (Anti-Rabbit IgG-HRP, Bio-Rad Laboratories, Hercules, CA, United States) for 1 h at RT. Protein levels were determined using enhanced chemiluminescence (Bio-Rad Laboratories, Hercules, CA, United States) and ChemiDoc XRS + (Bio-Rad Laboratories, Hercules, CA, United States).

Whole cell protein lysates were prepared using RIPA buffer containing 0.2% SDS, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF and EDTA-free mini-complete protease inhibitor cocktail tablet (Roche). Tissue samples or cells were homogenized in RIPA Lysis Buffer. The protein concentration was determined using Bradford’s Reagent (BioRad). 40 µg of protein was subjected to polyacrylamide gel electrophoresis and transferred on to a nitrocellulose membrane (Merck Millipore). Membranes were incubated with respective primary antibody dilutions prepared in Tris Buffered Saline (TBS)-Tween for 2 h at room temperature or overnight at 4 °C. Membranes were then washed in TBS-Tween and incubated with secondary antibodies anti-rabbit IgG–HRP (BioRad) or anti mouse IgG-HRP (BioRad) diluted (1:10.000) in TBS-Tween for 1 h at room temperature. Protein-antibody complexes were detected by Substrate Detection Kit (Thermofischer, USA). Quantification of signal was done via densitometry analysis, using the Image J software. Actin was used as loading control for whole cell lysates and Lamin A/C and GAPDH were used as loading controls for nuclear and cytoplasmic fractions respectively.