Blotto non fat dry milk

After 24 h IL-1β stimulation, F-NL cells were lysed with RIPA buffer and frozen at −80 °C. Proteins were purified with high speed centrifugation and their concentration was determined by bicinchoninic acid (BCA) assay (ThermoFisher, Waltham, MA, USA). Proteins were diluted with 4× Laemmli buffer and boiled for 10 min. 10–20 μg of total protein were separated with SDS-PAGE and transferred into PVDF membrane. After 1 h blocking with 5% Blotto non-fat dry milk (Santa Cruz, Active Santa Cruz, CA, USA) in TBS 0.05% tween 20, the membranes were incubated with specific antibodies recognizing human forms of COX-2 (160112, Cayman Chemical), GAPDH (sc-47724, Santa Cruz), α-Tubulin (sc-8035, Santa Cruz), α-SMA (ab5694, Abcam, Cambridge, UK), COL1 (ab34710, Abcam) and TIA-1 (sc-1751, Santa Cruz). The optical densitometry (OD) of the protein bands was analyzed using Image Lab™ Software (Bio-Rad, Hercules, CA, USA). Data were normalized with the loading control GAPDH or α-Tubulin and fold changes from control condition were calculated.

Protein extract preparation from tissue samples and western blotting were performed as described previously [21 (link)]. Primary antibodies against the following antigens were used: CACNA1C (guinea pig, 1:1000, Alomone Labs, Jerusalem, Israel), SRF (rabbit, 1:500, Santa Cruz Biotechnology, Dallas, TX), or GAPDH (rabbit, 1:1500, Cell Signaling Technology, Danvers, MA). All antibodies were incubated in 5% Blotto, non-fat dry milk (Santa Cruz Biotechnology, Dallas, TX). HRP-conjugated goat anti-guinea pig (AP108P), goat-anti-rabbit (AP307P), or goat anti-mouse (AP308P) secondary antibodies (1:50,000; EMD Millipore, Billerica, MA) were used with an enhanced chemiluminescence method (Amersham ECL Advantage, GE Healthcare Life Sciences, Pittsburgh, PA). The protein bands were captured using a CCD-camera system (EC3 410 Imaging System; UVP, Upland, CA) and analyzed with VisionWorksLS software (Version 6.8; UVP, Upland, CA). The expression level of CACNA1C and SRF was normalized to the ratio of CACNA1C and SRF area density divided by GAPDH area density. For quantitative measurements, the values of relative expression for CACNA1C and SRF in KO5D, KO10D, and KO15D small intestine were compared to the expression in WT10D small intestine (set to 1.0).

Total proteins from CR-CSphCs were isolated as previously described [17 (link)]. After performing SDS-PAGE, proteins were blotted on nitrocellulose membranes. Membranes were incubated with blocking solution (5% Blotto, nonfat dry milk, Santa Cruz Biotechnology, Dallas, TX, USA, PBS 0.1% Tween 20) for 45 min at R.T. and then exposed at 4 °C for 16 h with antibodies against phospho-AKT xp (Ser473; D9E, rabbit, IgG, CST, Danvers, MA, USA), AKT (rabbit polyclonal, CST, Danvers, MA, USA), phospho-MEK1/2 (Ser217/221; 41G9, rabbit IgG, CST, Danvers, MA, USA), MEK1/2 (rabbit polyclonal, CST, Danvers, MA, USA), phospho-ERK 1/2 (Thr202/Tyr204; rabbit polyclonal, CST, Danvers, MA, USA), ERK 1/2 (137F5, rabbit IgG, CST, Danvers, MA, USA) or Myc (rabbit polyclonal, CST, Danvers, MA, USA). After incubation with anti-mouse or anti-rabbit HRP-conjugated secondary antibody (goat IgG; Thermo Fisher Scientific, Waltham, MA, USA), chemiluminescence signals were detected with Amersham imager 600 (GE Healthcare, USA). The densitometric analysis of β-actin (8H10D10, mouse, CST, Danvers, MA, USA)-normalized protein levels was undertaken with ImageJ software.

Protein isolation was carried out using tissue protein extraction reagent (T-PER, Pierce, USA). Following centrifugation, resultant supernatant was collected and quantified using BCA kit, Pierce, USA. An equal amount of protein was resolved using 10 to 12% SDS-polyacrylamide gel. The resolved gel was then transferred to a polyvinyldine difluoride membrane (Thermo Scientific, Waltham, MA, USA). Blocking of the membrane was performed using 3–5% blotto, nonfat dry milk (Santa Cruz, Dallas, TX, USA) in TBS containing 0.1% Tween-20, at room temperature for 1 h. The membrane was then incubated with primary antibody at 4 °C overnight. Following three subsequent washings, the membrane was incubated with secondary anti-Rabbit (Cell signaling, Danvers, MA, USA. Cat No: 7074) or anti-mouse (Cell signaling, Danvers, MA, USA. Cat No: 7076) antibody for 1 h at room temperature. Blots were visualized using super signal chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA. Cat No: 34080). SIRT-3 antibody (Cell signaling, Danvers, MA, USA. Cat No: 5490), SIRT-1 antibody (Abcam, Cambridge, MA, USA. Cat No: ab157401), TFAM antibody (Abcam, Cambridge, MA, USA. Cat No: ab131607), GAPDH antibody (Cell signaling, Danvers, MA, USA. Cat No: 2118), and anti-acetylated lysine antibody (Cell signaling, Danvers, MA, USA. Cat No: 9441) were used for the study.

For lectin-blot, the PVDF membrane was blocked with T-PBS (PBS + 0.1% v/v Tween), 3% BSA for 1 h at room temperature, incubated with ALL 1.1 µg/ml in PBS overnight (4°C) and washed 3 times with T-PBS followed by incubation with Streptavidin HRP (Strp-HRP, 1:7000; Vector Laboratories) in T-PBS, 5% Blotto-non-fat dry milk (Santa Cruz Biotechnology) for 1 h at room temperature and a final wash with T-PBS, 0.2% Triton X-100. Western-blot membranes were blocked for 1 h at room temperature using T-TBS (Tris-buffered saline + 0.1% Tween), 3% BSA, followed by incubation with anti-moesin HRP (EP1863Y, 1:5000; Abcam) diluted in TBS overnight at 4°C and washed with T-TBS, 0.2% Triton X-100. Both membranes were developed using Immobilon Western Chemilum HRP Substrate (Merck) on autoradiographic Kodak Biomax films (Sigma-Aldrich), and data were analyzed using Lab 6.0.1 software (Bio-Rad).

Proteins were transferred onto nitrocellulose membranes using Trans-Blot Transfer System (Bio-Rad) and total protein on the membrane was imaged using the ChemiDoc Imaging System (Bio-Rad). Membranes were blocked in 5% Blotto non-fat dry milk (Santa Cruz Biotech, sc2325) in 1× TBS-T (10 mM Tris-HCl, 0.15 M NaCl, 0.05% Tween 20) over night at 4°C. Incubation with 1/500 goat anti-CAPZB (GeneTex, GTX89785) and 1/1,000 rabbit anti-PALM (Abcam, ab234739) primary antibodies was performed at room temperature for 2 h and then washed three times in TBS-T. Membranes were incubated first with 1/15,000 680RD donkey anti-rabbit (LI-COR Biosciences, 925-68073) for 1 h at room temperature, washed three times and imaged using the Odyssey^® CLx Imaging System (LI-COR) to detect PALM signal. To detect CAPZB signal, we incubated the membranes with 1/5,000 donkey anti-goat IgG conjugated to HRP (Jackson ImmunoResearch, 705-035-147), washed three times and develop using the Clarity Western ECL Substrate (Bio-Rad, 1705061) in the ChemiDoc MP Imaging System (Bio-Rad, 17001402).