Dc protein kit

Liver tissues were resuspended in RIPA lysis buffer. Lysates were centrifuged for 15 min at 12000 g and 4 °C, and protein contents of the supernatant were determined using DC protein kit (Bio-Rad Laboratories, CA, USA). Aliquots of the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to PVDF membranes (Bio-Rad Laboratories, CA, USA). PVDF membranes were incubated with antibodies against phospho-Eif2α, GRP78, CHOP, ATF6 (90KD), ATF6 (50KD), shear-XBP1 (XBP1-s), unshear-XBP1 (XBP1-u), Cleaved-Capsase3 (Cleaved-Casp3), phospho-IRS1 (Ser307), phospho-Akt (Ser473), IRS1, Akt, phospho-NFκB-p65, phospho-IκBα, phospho- JNK, phospho-p38-MAPK, NFκB-p65, IκBα, JNK1, p38-MAPK and β-actin. All antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Proteins were extracted from cells with a lysis buffer (NP40 1%, SDS 0.1%) and quantified with the Dc protein kit (Biorad). Forty μg of total proteins were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. We used the following primary antibodies: anti-SF3B1 (1:5000, #170854 Abcam), anti-FLAG (1:5000, #F3165, Sigma), anti-GAPDH (1:5000), and anti-b-actin (1:1000, #ab8226, Abcam). Donkey anti-mouse (1:5000, #926-32222, LI-COR) and goat anti-rabbit (1:5000, #926-32211, LI-COR) secondary antibodies were used. Immunofluorescence was detected using Odyssey Infrared Imaging system (LI-COR). Quantification was performed with Image Studio Lite software. β-actin or GAPDH were used as references to normalize quantification.

The secretomes were obtained following a protocol adapted from Fong et al. [44 (link)] and previously described by our group [7 (link)]. In summary, DSCs or ASCs monolayers at 90% confluence were subjected to a triple wash with PBS and maintained in DMEM without FBS for 48 h. Cell supernatants were collected and processed by centrifugation (5 min, 300 g) to pellet and exclude eventual floating dead cells, and then filtered through a 0.22 µm filter to remove debris. The resulting secretome was concentrated tenfold using an Amicon Ultra-15-Centrifugal Filter 3 kDa MWCO (Millipore) by centrifugation (45 min, 5,000 g), following the manufacturer's instructions. The concentrated secretome was aliquoted and stored at − 80 °C for up to 4 months for downstream applications. The total protein content of the secretome was quantified using the DC Protein kit (Bio-Rad) according to the manufacturer’s protocol, and reading was carried out using the Tecan Infinite M200 microplate reader. Total protein secreted was similar in seDSC and seASC (average of 194.4 and 209.4 µg per 10⁶ cells, respectively). Secretomes were diluted to a final concentration of 1 µg/µl and wound healing studies used a dosage of 200 µg of total protein per wound, in reference to cell group controls that received 10⁶ DSC or ASC per wound.

The assay was conducted following the procedure as described previously by Hannan et al. [27 (link)]. The ethanol extract of B. monosperma (250, 500, and 1000 mg/kg) was administered by gastric gavage to 20 hr fasted nondiabetic rats. After 60 min, the rats were sacrificed and the small intestine was isolated, cut longitudinally, rinsed with ice-cold saline, and homogenized in 10 mL saline (0.9% NaCl). Aliquots of homogenate were incubated with 40 mM sucrose at 37°C for 60 min. The amount of protein was determined by DC Protein Kit (Bio Rad, USA). Disaccharidase activity was determined from the glucose concentration converted from sucrose as μmol/mg protein/h.