Westernbright quantum hrp substrate

Sample preparation and western blotting were performed as previously described [21 (link)]. Cells, including detached ones, were freshly lysed in cold caspase lysis buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂ and 1% NP-40) reconstituted with 0.25 mM AEBSF and Halt Protease and Phosphatase Inhibitor Cocktail (78440, Thermo Scientific). Cell debris was removed by high-speed centrifugation for 20 min at and 4 °C, and protein concentration was quantified with Pierce BCA Protein Assay Kit (23225, Thermo Scientific). Briefly, 25 μg of protein were resolved on Bolt 4–12% Bis-Tris Plus Gels (NW04125BOX, Invitrogen) under reducing conditions according to manufacturer’s instructions. After transfer onto PVDF membranes (IPVH00010, Millipore) using Criterion blotter (1704070, Bio-Rad), blocked membranes were incubated overnight at 4 °C with the indicated primary antibodies, washed and stained with secondary antibodies for 1 h at RT. Images were detected with WesternBright Quantum HRP substrate (K-12042-D10, Advansta) on LAS-3000 Imager (Fujifilm) and processed using ImageJ software with brightness/contrast adjustment applied to an entirely digital image if necessary.

Five microlitre CSF was diluted in 15 μl PBS containing a complete protease inhibitor (Thermo Scientific). The samples were denatured in 5× Laemmli buffer, separated by SDS–PAGE gel electrophoresis, and transferred to the nitrocellulose membrane. Following blocking with Odyssey Blocking buffer (LI‐COR Biosciences), the membrane was first incubated with biotinylated goat anti‐mouse IgG (1:2,000; Thermo Scientific) and then with HRP‐conjugated Streptavidin (1:5,000; Thermo Scientific). Protein bands were detected and quantified by WesternBright Quantum HRP substrate (Advansta) in Amersham Imager 600 (GE Healthcare).

Cells were heated at 100°C in Laemmli loading buffer (4% SDS, 20% glycerol, 120 mM Tris-HCl, pH 6.8, and 0.2% bromophenol blue) for 5 min. Protein from 10⁶ cells/lane were separated on a 10% SDS-PAGE gel, followed by a transfer to a polyvinylidene difluoride membrane using the Mini Trans-Blot system (Bio-Rad), according to the manufacturer’s protocol. The transferred membrane was blocked with 5% skimmed milk (Sigma-Aldrich) in TBS-T (50 mM Tris-HCl, 100 mM NaCl, pH 7.6, and 0.1% Tween-20) for 1 h. Mouse monoclonal antibodies: anti-TY (1:200; Bastin et al., 1996 (link)), L8C4, L13D6 (Kohl et al., 1999 (link); 1:200), a kind gift of Professor Keith Gull (University of Oxford, Oxford, UK) were incubated with the membrane for 1 h. Three membrane washes were performed with TBS-T and incubated for 1 h with anti-mouse IgG coupled to HRP (Jackson Laboratory), diluted 1:10,000. The membrane was washed three times with TBS-T following secondary antibody incubation. For final detection of protein, the chemiluminescence reaction was performed with WesternBright Quantum HRP substrate (Advansta) according to the manufacturer’s protocol.

To quantify HC1 and HC2 protein expression, first instar larvae dissected from the uteri were homogenized twice in ice-cold lysis buffer which contained 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5% Na-deoxycholate, 0.5% NP-40, 0.5% SDS, and protease inhibitor (EDTA-free). Lysates were cleared by centrifugation at 15.000×g at 4 °C for 30 min, and the protein content was determined by a protein assay using bovine serum albumin (BSA) as a standard (Bradford method). Sixty micrograms of protein from each sample was reconstituted directly in the appropriate amount of sample buffer and separated in Mini-Protean TGX System Precast Protein Gels (Bio-Rad, Hercules, CA, USA) and transferred to Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad, Hercules, CA, USA). The membranes were washed and blocked in 0.02 M Tris-buffered saline containing 5% BSA and 0.1% Tween 20 and then incubated overnight at 4 °C with anti-HC1 or HC2 antibodies diluted 1:500. Next, the membranes were washed with TBST (Tris-buffered saline containing 0.1% Tween 20) and incubated for 1 h with a horseradish peroxidase-conjugated goat anti-rabbit antibody (Santa Cruz, USA) diluted 1:1000. Signals were detected by chemiluminescence using WesternBright Quantum HRP substrate (Advansta Inc., Menlo Park, USA) and visualized using the Chemidoc™ XRS + System (Bio-Rad, Hercules, USA).

Western blot analysis is a commonly used technique to quantify specific proteins. To prepare total cellular protein extracts, cells were seeded in 6 cm dishes at a maximum confluence of 70%. After 24 h, the total protein extracts were collected by scraping the cells in RIPA buffer (Eurx, Poland, and 1 mM PMSF) supplemented with a mixture of 1x protease inhibitors (Roche Molecular Systems, Inc; Rotkreuz, Switzerland). The samples were incubated on ice for 15 min, and the lysates were centrifuged (4 °C for 20 min at 22,000× g). Protein concentration was determined using a Protein Assay Kit (Bio-Rad; Hercules, CA, USA) according to the manufacturer’s instructions. Equal amounts of protein, for each sample were separated by SDS-PAGE on 12% polyacrylamide separating gels and transferred to a nitrocellulose membrane using a Trans Blot Turbo system (Bio-Rad; Hercules, CA, USA) for 10 min. Membranes were blocked in 5% skim milk/TTBS (0.25 M Tris–HCl (pH 7.5), 0.15 M NaCl, and 0.1% Tween-20) for 1 h. Antibody–antigen interactions were detected using secondary antibodies (Table 2) and visualized using WesternBright Quantum HRP substrate (Advansta; San Jose, CA, USA). X-ray films (Carestream Health, Inc, Rochester, NY, USA) were used to detect chemiluminescent signals. β-Actin was used as a protein control.

Frozen kidney and liver tissues were crushed in liquid nitrogen using a Mixer Mill during 30 s at 30 Hz (MM400, Retsch technology), and 12 mg of tissue powder were lysed with 500 µL of RIPA solution (Ref#R0278, Sigma-Aldrich). The protein extracts were centrifuged for 20 min at 16,000 g and 4 °C, and protein concentration in the supernatant was determined using the BCA protein assay Kit (Ref#K812-1000, Biovision). Each protein sample (45 µg) was denatured and reduced in Laemmli buffer (62.5 mM TRIS pH 6.8, 2% SDS; 10% glycerol) containing 5% ß-mercaptoethanol before SDS-PAGE on a 4–15% polyacrylamide gel (Bio-Rad). Proteins were transferred onto a nitrocellulose membrane blocked with AdvanBlock-Chemi blocking solution (Advansta) during 1 h at room temperature. After washing in PBS-0.1% Tween 20, the membrane was incubated overnight at 4 °C with a rabbit polyclonal anti-MMUT primary antibody at 1/1000 in blocking solution (MUT Rabbit pAb, Ref#A3969, ABclonal). Revelation of the primary antibodies (after washing) was performed by incubation with goat anti-rabbit Horseradish peroxidase-conjugated secondary antibody (Ref#A0545, Sigma-Aldrick) at 1/10000 ratio in blocking solution for 1 h at room temperature, followed by enhanced chemiluminescence detection (WesternBright Quantum HRP substrate, Advansta) on a ChemiDoc Touch low-light camera (Bio-Rad) in automatic mode.