Goat anti rabbit hrp

Cell pellets from patient and control fibroblasts were lysed using RIPA buffer and Protein Inhibitor Cocktail on ice, and protein levels were measured using a Bradford Assay. Twenty micrograms of protein was run on a 12% SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane. Membranes were incubated overnight at 4 °C, with mouse anti-Drp1 (Abcam; 1:1000), mouse anti-OPA1 (BD Biosciences; 1:1000), mouse anti-Mfn1 (Abcam; 1:1000) or rabbit anti-Mfn2 (St Johns Laboratory; 1:500). Membranes were also probed for β actin as a loading control (St Johns Laboratory; 1:1000). Membranes were then incubated with goat anti-mouse HRP (Abcam; 1:10,000) or goat anti-rabbit HRP (Dako; 1:5000), as appropriate. Membranes were imaged using the G box chemi system using GeneSnap software (Syngene). Densitometry was analyzed using GeneTools software (Syngene).

Cell pellets were lysed in 50 mM Tris pH 7.9/8 M Urea/1%Chaps and incubated at 4 °C with agitation for at least 30 min. The lysate was cleared by centrifugation and the supernatant was collected. The protein concentration of the extracts was measured by Bradford assay. About 25 μg of supernatants were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto a Hybond-C Extra Nitrocellulose membrane (Fisher Scientific UK, Loughborough, UK). The membrane was blocked in 5% milk, 0.1% Tween-20 in TBS buffer for 1 h and probed overnight with antibodies against BRG1 (Santa Cruz, sc-17796, dilution 1:2000), a-tubulin (Abcam Ab7291, dilution 1:5000), p21 (Cell Signalling, 2947 S, dilution 1:2000), INO80 (Bethyl, A303-371A, dilution 1:2000) in 5% milk, 0.1% Tween-20 in TBS buffer. The membrane was washed three times with 0.1% Tween-20 in TBS and incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies in 5% milk, 0.1% Tween-20 in TBS. The secondary antibodies used were Rabbit anti-Mouse HRP (Dako, P0260, dilution 1:5000) and Goat anti-Rabbit HRP (Dako, P0448, dilution 1:5000). Proteins were visualised by using in-house ECL reagent or SuperSignal West Pico Chemiluminescent Substrate (Life Technologies).

Protein samples corresponding to 15 whole flies were prepared with RIPA buffer, supplemented with protease inhibitor cocktail (Roche). 50μg of total lysate as determined by Bradford assay (Sigma) was loaded and separated on a 10% acrylamide precast Novex gel (Invitrogen) under reducing conditions and transferred onto nitrocellulose membrane. Primary antibodies were incubated at 4°C overnight. Subsequently, species-specific HRP-conjugated secondary antibodies were used. Primary antibodies used are as follows: rabbit anti-P-Smad2 (Cell Signaling Technology, #3108) 1:1000; mouse anti-β-tubulin antibody 1:10 000 (Cell Signaling Technology, #4054). Secondary antibodies used were: goat anti-rabbit -HRP 1:5000 (Dako); anti-mouse-HRP 1:5000 (GE Healthcare). Blots were visualized by chemiluminescence using ECL (GE Healthcare) or Chemiluminescent Peroxidase Substrate-1 (Sigma).

Ganetespib was obtained by Synta Pharmaceuticals (Lexington, MA, USA). ABT737 and ABT199 were kindly donated by Dr Vogler (University of Leicester, Leicester, UK). PU-H71 and Radicicol were purchased from Tocris Bioscience (Bristol, UK). 17-AAG was purchased from Sigma (St. Louis, MO, USA). The antibodies against PARP, BID, BIK, PUMA, CASPASE 8, BCL-2, BCL-Xl, BCL-w and GFP were obtained from Cell Signaling (Danvers, MA, USA), BAX, BAK and MCL1 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and β-tubulin was obtained from Abcam (Cambridge, UK). Cytochrome-c antibody was purchased from BD PharMingen (Oxford, UK). Secondary antibodies were goat anti-rabbit HRP (DAKO, Glostrup, Denmark) and donkey anti-mouse HRP (GE Healthcare, Amersham, UK).

The transfected NG108-15 cells were harvested in the lysis buffer (150 mM NaCl, 1.0% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 10 mM NaF, 1 mM Na₃VO₄, 1.0% NP-40, and 40 mM Tris-HCl, pH = 8.0) supplemented with protease inhibitor (Cat# 469311001, Roche) on ice. The samples were boiled at 100°C for 30 min and then the supernatants were loaded in 10% SDS-PAGE gel for gel electrophoresis. The proteins on the gels were transferred onto the PVDF membranes (Cat# 88520, Thermo Fisher Scientific) and these membranes were blocked with 5% nonfat dry milk. The membranes were incubated with the corresponding primary antibodies 1:5000 for α-Tubulin (Cat# T8203, Sigma); 1:500 for TRPC1 (Cat# ACC-101, Alomone Labs)/5 (Cat# ACC-020, Alomone Labs)/6 (Cat#ACC-120, Alomone Labs) and 1:200 for TMEM16F (Cat# ACL-016, Alomone Labs) at 4°C overnight with gentle shaking. Secondary antibodies Goat-anti-rabbit HRP (Cat# P0448, Dako) and Goat-anti-mouse (Cat# P0447, Dako) 1:5000 in 5% BSA were used. Membranes were washed with PBST three times and developed with an ECL-HRP (Cat# WBULS0100, Millipore) system. The gray value ratios of protein bands were quantified using ImageJ software.

Rabbit anti-vimentin antibody (ab16700), rabbit anti-tubulin (ab18251), mouse anti-LAMP1 (ab25630) were from (Abcam, Cambridge, UK). Mouse anti-tubulin (T5168, Sigma-Aldrich), rabbit anti-LC3 (0260–100 LC3-2G6, Nanotools, Teningen, Germany), rpS6 (2317) and phosphorylated rpS6 (p-rpS6 S235/236, 4856) were from (New England Biolabs, Herts, UK). Secondary antibodies for immunostaining: anti-rabbit FITC and anti-rabbit TRIC were from (Sigma-Aldrich), anti-rabbit Alexa Fluor 647 (A21246, ThermoFisher, Paisley, UK), anti-rabbit Alexa Fluor 488 (A11034, Invitrogen), anti-mouse Alexa Fluor 568 (A11004, Invitrogen,). Actin staining was performed using Alexa Fluor 594 Phalloidin (A12381, Invitrogen,) or Alexa Fluor 488 Phalloidin (A12379, Invitrogen,). LysoTracker Red (L7528, ThermoFisher) was used to visualise lysosomes in live cell imaging. Secondary antibodies for western immunoblotting: goat anti-rabbit HRP (P0448, Dako, Santa Clara. USA) and goat anti-mouse HRP (P0447, Dako).

Choroid plexus was dissected from the brain of NBCe2 KO and WT mice and transferred to sample buffer (0.3 m sucrose, 25 mm imidazole, 1 mm ethylenediaminetetraacetic acid, 0.1 m sodium dodecyl sulphate, and 0.04 m dithiothreitol, Bromophenol Blue, pH 6.8). Samples were sonicated by 5 bursts 3 times at 60% using a Model 150 V/T sonicator (BioLogics Inc., Cary, NC, USA) and heated for 15 min at 65°C. Samples were loaded on 4–12% polyacrylamide SDS gels and separated by electrophoresis. After transfer to a polyvinylidene difluoride membrane (PVDF, Ambion, Foster City, CA, USA), the membrane was blocked with 5% skimmed milk in PBS‐T (PBS with 0.1% vol/vol Tween). The membrane was incubated with the primary antibody in 1% BSA, 2 mm NaN₃ in PBS‐T overnight at 4°C. After washing, the membrane was incubated with secondary antibody (goat anti‐rabbit HRP, 1:3000, Dako, Glostrup, Denmark) for 1 h at room temperature. ECL Plus (GE Healthcare) was used for visualization of immunoreactive bands using an ImageQuant LAS4000 (GE Healthcare, Chicago, IL, USA) chemiluminescence digital analyser.