Imagequant las 4000 mini

Cells were washed with cold PBS and resuspended in lysis buffer containing 25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, in the presence of HaltProtease Inhibitor Cocktail, EDTA-Free (Thermo) and incubated on ice for 20 min. Lysates were sonicated for 10 min at 40 kHz in a bath sonicator (Emag Emmi-D280) and centrifuged at 18,000×g for 10 min at 4 °C. Total protein concentration was determined using Roti-Quant kit (Carl Roth) according to the manufacturer’s instructions. Lysates were diluted in Roti-Load (Carl Roth) and heated at 95 °C for 5 min. Then, 15–35 µg protein was loaded and separated by SDS-PAGE in 4–20% Mini-Protean TGX Precast gels (Bio-Rad). Gels were transferred to PVDF membranes using the iBlot system (Thermo). Membranes were blocked for 1 h in 5% low-fat powdered milk/1% Tween/TBS (TBS-T), washed and incubated overnight in primary antibodies at 4 °C and for 1 h at RT in HRP-conjugated secondary antibodies, all diluted in 3% milk/TBS-T. Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and the ImageQuant LAS 4000 mini. Tubulin was used as an internal loading control, and the relative intensities of protein bands were quantified using ImageJ and Excel.

Brains were extracted from four animals for each condition (control, p110α^nKO, and p110β^nKO). Hippocampi were dissected and homogenized in a glass-teflon potter on ice-cold buffer containing 20 mM Hepes, 0.1% NP-40, cOmplete mini EDTA-free, and phosSTOP and centrifuged at 13,000g for 10 min at 4°C. The supernatant processed on SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Immoblot-P Millipore). After membrane blocking for 1 hour in 5% skim milk in Tris-buffered saline with 0.1% Tween 20, primary antibodies were incubated overnight at 4°C in blocking solution. Membranes were washed in TBST and incubated for 1 hour in the corresponding secondary antibodies (peroxidase-conjugated anti-rabbit or anti-mouse; the Jackson ImmunoResearch, 711-035-152 and 715-035-151). Detection was carried out by chemiluminescence (Immobilon Western, Millipore) using ImageQuant LAS 4000 mini biomolecular imager.

Total protein was extracted from the shLuc and shMTA2 cells. Protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA, USA). Primary antibodies, namely MTA2, MMP-9, and β-actin, were incubated overnight at 4 °C, washed twice, and then incubated with secondary antirabbit and antimouse IgG for 60 min. Immunoreactive bands were detected with a chemiluminescence kit (Millipore, Billerica, MA, USA) using the ImageQuant LAS 4000 mini according to the manufacturer’s instructions.

Cells were washed twice with PBS and lysed on ice with Radio-Immunoprecipitation Assay (RIPA) buffer (Thermo Fisher, Waltham, MA, USA) containing 1% protease inhibitor cocktail (Sigma). Protein concentration was determined using bicinchoninic acid (BCA) protein quantitative analysis kit (Beyotime, Shanghai, China). Proteins were loaded on a 6%-12% SDS-PAGE, and transferred to polyvinyl difluoride (PVDF) membranes (Sigma-Aldrich). The membranes were incubated at 4℃ overnight with the primary antibodies for E-cadherin (1:1000, Cell Signaling Technology), α-SMA (1:1000, Abcam), LOX (1:1000, Abcam), SM-MHC (1:1000, Abcam), desmin (1:1000, Abcam), GAPDH (1:1000, Beyotime) and β-tubulin (1:1000, Cell Signaling Technology). The membranes were incubated with HRP labeled secondary antibodies for 1 h at room temperature, and the signals were developed with enhanced chemiluminescence (ECL) reagents (Millipore, Burlington, MA, USA) and digitized on Image Quant LAS 4000 mini. Image quantification was carried out with Quantity One software (Bio-Rad, Hercules, California, USA).

Cells treated with DMSO, Niebla sp. (1), or tumidulin for 48 h were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer [24 (link)]. Antibodies against ALDH1, CD44, CD133, Lgr5, Msi1, and α-tubulin were used as previously described [17 (link)]. Antibodies against Gli1 (sc-20687; SANTA CRUZ, Dallas, TX, USA), Gli2 (sc-271786; SANTA CRUZ), Smoothened (SMO; ab72130; Abcam, Cambridge, MA, USA), and β-Actin (sc-47778; SANTA CRUZ) were detected with horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) using an Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore, Billerica, MA, USA) and luminescence imaging on an Image Quant LAS 4000 mini. Bands were measured using Multi-Gauge 3.0 software, and the relative density was calculated based on the density of the β-actin bands in each sample. Values are expressed as arbitrary densitometric units corresponding to signal intensity.

Cells treated with UA or KU for 24 h were washed twice with ice-cold PBS and lysed in lysisbuffer^{26 (link)}. Antibodies against E-cadherin (61018, BD Biosciences, San Diego, CA, USA), N-cadherin (610921, BD Biosciences), Snail/Slug (ab180714, Abcam, Cambridge, MA, USA), Twist (ab49254, Abcam), PARP (9542, Cell Signaling), Caspase-3 (9662, Cell Signaling), α-Tubulin (2125, Cell Signaling), and ZEB2 (HPA003456, Sigma) were detected with horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) with the use of Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore, Billerica, MA, USA) and luminescence imaging (Image Quant LAS 4000 mini). Multi-Gauge 3.0 was used to measure bands, and relative density was calculated based on the density of the α-Tubulin bands in each sample. Expression of values were as arbitrary densitometric units corresponding to signal intensity.