Amersham ecl rabbit igg

All AQP4 mutant constructs were studied for OAP assembly by using blue native gel electrophoresis (BN-PAGE). Therefore, HEK-293A cells were transfected as described before. Cells were lysed in 1X NativePage^TM Sample Buffer (Invitrogen, # BN2003) supplemented with 500 mM 6-aminohexanoic acid, 1 % Triton X-100 and protease inhibitor cocktail, incubated for 30 min on ice and centrifuged at 22,000×g for 30 min. Supernatants were supplemented with NativePage^TM 5 % G-250 (Invitrogen, #BN2004) and loaded to NativePAGE™ Novex™ 3-12 % Bis-Tris Protein Gels (Invitrogen, # BN1001). The running buffers were prepared according to the manufacturer’s protocol. Proteins were blotted onto polyvinylidene difluoride (PVDF) membrane using NuPage Transfer Buffer (Invitrogen, #NP0006). For immunoblot analysis, proteins were fixed for 15 min in 8 % acetic acid and membranes were blocked in 1 % Amersham ECL Prime Blocking Reagent (GE Healthcare, # RPN418) diluted in 0.1 % PBS-Tween. Primary rabbit anti-AQP4 antibody (Sigma, # A5971) was incubated at 4 °C overnight. Secondary antibody Amersham ECL Rabbit IgG (GE Healthcare, # Na934) was incubated at RT for 1 h. Labeled proteins were detected using the WesternBright ECL (Biozym, # 541004) and visualized on the Fusion FX7 Vilber Lourmat imaging system.

Western blot was carried out to analyze activation-states of the Hh- and PI3K/AKT/mTOR pathways. Tumor tissue samples from the same animals used in the gene expression analysis were homogenized in RIPA Lysis and Extraction Buffer (Thermo Scientific) using the TissueLyser LT (Qiagen) and Bioruptor® (Diagenode). Cell debris was removed by centrifugation and the protein extract was stored at −20 °C. Protein extracts (100 μg) were run on SDS-PAGE using Mini-PROTEAN® TGX™ Precast Gels (Bio-Rad) and transferred to nitrocellulose membranes using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). Antibodies specific to GLI1 (ab151796, Abcam), GLI2 (LS-C313075, LifeSpan BioSciences), S6 (#2217, Cell Signaling Technology), AKT (#9272, Cell Signaling Technology), p-AKT (#9271, Cell Signaling Technology) and GAPDH (ab9485, Abcam, used as control) were detected using Amersham ECL Rabbit IgG (NA934VS, GE Healthcare Life Sciences). SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific) was used for detection and digitalized images were acquired using Fujifilm Luminescent Image Analyzer LAS-1000 (Fujifilm, Tokyo, Japan).

Samples derived from whole cell lysates were separated by SDS–PAGE and transferred to a nitrocellulose membrane for immunoblotting. Blots were probed using the following primary antibodies: rabbit anti-CENP-C (1.7 μg ml⁻¹) (ref. 54 (link)), mouse mAb anti-α-tubulin (1:4,000, Sigma-Aldrich #T9026), or human anti-centromere antibodies (2 μg ml⁻¹, Antibodies Incorporated #15-235). The blots were subsequently probed using the following horseradish peroxidase-conjugated secondary antibodies: Donkey Anti-Human IgG (1:10,000, Jackson ImmunoResearch Laboratories #709-035-149), Amersham ECL Mouse IgG (1:2,000, GE Life Sciences #NA931), Amersham ECL Rabbit IgG (1:2,000, GE Life Sciences #NA934V). Antibodies were detected by enhanced chemiluminescence (Thermo Scientific). Please refer to Supplementary Fig. 9 for the uncropped blots of Fig. 1c and Supplementary Fig. 1d. Note that Supplementary Fig. 1j is not cropped.