Notch3

Adipose tissue or cell samples were homogenized and total protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific Inc., Billerica, MA). Equal amounts of proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore Bedford, MA). The membranes were cut according to the molecular weight and then incubated with antibodies directed against total and cleaved Notch1 (Santa Cruz Biotechnology, Santa Cruz, CA; dilution, 1:1000, Cell Signaling Technology, MA; dilation, 1:1000), Notch3 (Proteintech, Chicago, IL; dilution, 1:1000), Pref-1 (Abcam, Cambridge, UK), respectively. Then, the membranes were further incubated with HRP-linked secondary antibody (dilution, 1:2000) at room temperature for 1 h. After washing with TBS-T three times, protein expression was visualized using the enhanced Chemi-Lumi one system (Nacalai Tesque, Kyoto, Japan). The intensity of protein bands was normalized to the amount of β-actin (an internal control, Cell Signaling Technology, MA; dilution, 1:2000) and expressed as ratio (fold increase) of the control value.

Formalin-fixed paraffin-embedded gallbladder tissues were processed for histology and immunohistochemistry by standard procedures. Primary antibodies used for immunostaining were: Notch1 (Cell Signaling, No. 3608, 1:100), Notch2 (DSHB, University of Iowa, C651.6DbHN, 1:200), Notch3 (ProteinTech, 55114-1-AP, 1:100), Notch4 (Millipore, 09-089, 1:100), and Jagged1 (Santa Cruz, sc-6011, 1:100). X-Gal staining was performed as previously described [22 (link)]. For Western blot analysis, gallbladder tissues were lysed in RIPA buffer (Boston BioProducts) supplemented with protease inhibitor (Roche), and processed according to standard methodology. Antibodies for probing specified proteins are as follows: Notch1, Notch2, Notch3, Notch4 and Jagged1 are the same as above (all with 1:1000 dilution), Hes1 (Millipore, AB5702, 1:500), and β-Actin (Santa Cruz, sc-81178, 1:1000).

Western blotting analysis was performed as described previously [99 (link),100 (link)]. Cells were briefly lysed with 1× Laemmli Sample Buffer solution (BioRad, Hercules, CA, USA, 1610737), boiled for 5 min to 95 °C, and sonicated. Lysates were separated on 4–20% Mini-PROTEAN^® TGX™ Precast Protein Gels (BioRad, 4561094) and transferred onto a PVDF membrane (BioRad, 1704272) using a semi-dry method of transfer (Bio-Rad Trans-blot Turbo system). The transferred blots were probed overnight with one of the following primary antibodies: rabbit anti-H3K4Me2 antibody (Diagenode, Denville, NJ, USA, C15200151), H3K27Ac (Diagenode, C15410196), rabbit anti-β-ACTIN (Li-Cor, 926-42210), JAG2 (Cell Signaling, #2205, C83A8), NOTCH3 (Proteintech, Chicago, IL, USA, 55114-1-AP), or NOTCH4 (Abcam, Cambridge, MA, USA, ab184742) primary antibodies. After incubation with IRDye 800 goat anti-rabbit (Li-Cor, Lincoln, NE, USA, 925-32211) and IRDye 680 goat anti-rabbit (Li-Cor, 925-68071) secondary antibodies (1:10,000) in 10% adult bovine serum blocking buffer was placed on a rocker for 1 h at room temperature. After several washes in 1XTBST, the protein signal was visualized on an Odyssey imaging system (Li-Cor, Lincoln, NE, USA) and quantified using β-actin as a normalizer with the optical density (OD) function of Image J software (NIH, Bethesda, MD, USA).

Proteins were extracted using RIPA buffer (Beyotime, Zhejiang, China) including protease inhibitor cocktail tablets (Roche Applied Science, Mannheim, Germany). Subsequently, the extracted proteins were separated by SDS-PAGE (Invitrogen), and transferred to PVDF membranes. The membranes were then incubated with the following primary antibodies: Ryr2 (ab302716, Abcam), Atp2a2 (#9580, Cell signaling technology, USA), Pln (MA3-922, Invitrogen), Notch3 (55114-1-AP, Proteintech), Phospho-Smad2/3 (#8828, CST), Phospho-Stat3 (#9145, CST), Stat3 (sc-8019, Santa cruz), and β-Actin (sc-47778, Santa cruz). Following washing, the blots were treated with IRDye 800 conjugated secondary antibodies (072‐07‐15‐06 or 072‐07‐18‐06, LI-COR Biosciences, Lincoln, NE, USA) at room temperature for 1 h. Images were captured using the Odyssey infrared imaging system and analyzed with the Odyssey Application Software v2 (LI-COR Biosciences).

The total protein in a sample was collected with RNA lysate and subsequently quantified and denatured; proteins were separated by SDS‐PAGE and transferred to Hybond membrane (Amersham), followed by blocking for 1 hour with 3% bovine serum albumen. Then incubated with primary antibodies against NOTCH3 (1:1000; Proteintech, Proteintech Group) for 2 hours, washed with TBST buffer 3 times, incubated with HRP‐conjugated anti‐rabbit secondary antibodies for 2 hours, washed three times and visualized using ECL luminescence solution.