Rabbit anti gapdh

For protein extraction from S2R+ cells, cell lysates were extracted in lysis buffer (50 mM TrisCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). For protein extraction from adult flies, one-day-old flies were collected, and proteins were extracted in lysis buffer with protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). Protein concentrations of the cleared lysate after two times centrifugation (13,000 r.p.m.) at 4 °C were measured, and the same amount of proteins was used for western blot analysis.
For immunostaining of western blots, following antibodies were used: mouse anti-V5 (1:5000, Invitrogen R960-25), mouse anti-Myc (1:1000, Santa Cruz sc-47694), rabbit anti-CCT4 (1:20,000) (this study), rat anti-CCT1 antibody (1:1000, Abcam ab90357), rat anti-Rheb (1:500) [48 (link)], and mouse S6K (1:1000) [40 (link)], rabbit phospho-S6K (1:1000, Cell signaling 9209), rabbit phospho-S6 (1:5000) [49 (link)], rabbit anti-Histone 3 (1:10,000, Millipore 05-928), rabbit anti-GAPDH (1:5000, GeneTex 100118), rabbit anti-Akt (1:1000, Cell Signaling 4691), rabbit anti-phospho-Akt (1:1000, Cell Signaling 4054), mouse anti-Tubulin (1:5000, Sigma T5168), guinea pig anti-TOR (1:2000) [50 (link)], rat anti-TOR (1:2000) [51 (link)].

The following primary antibodies were used: anti-AQP2 (7661AP) was kindly provided by Prof. Sebastian Frische; immunofluorescence (IF) and immunoblotting (IB) 1:1,000; rabbit anti-Ki67 (#4203–1, Epitomics, Cambridge, United Kingdom) IF 1:500; anti-B1/2 H⁺ATPase (#sc-55544, Santa Cruz, Dallas, TX, United States) IF and IB 1:1,000; rabbit anti-NKCC2 (#AB3562P, Millipore, Dramstad, Germany) IF and IB 1:1,000; rabbit anti-β1 integrin (#04-1109, Millipore, Dramstad Germany) IB 1:1,000; rabbit anti-ENaC alpha (SPC-403, StressMarq, Victoria, BC, Canada) IB 1:1000; mouse anti-β-Actin (#A2066 Sigma, Milan, Italy) IB 1:20,000; rabbit anti-GAPDH (#10018 GeneTex, Hsinchu City, Taiwan) IB 1:20,000. The following secondary antibodies were used: goat anti mouse Alexa Fluor 488 IF 1/800 (Invitrogen, CA, United States) goat anti-rabbit Cy3 (A10520, Invitrogen, CA, United States) IF 1:500; anti-mouse HRP conjugated (NA931V GE Healthcare, Little Chalfont, United Kingdom) IB 1:2,000; anti-rabbit HRP conjugated (NA934V GE Healthcare, Little Chalfont, United Kingdom) IB 1:2,000.

After cells were transfected with the indicated siRNA duplexes for 42 h, cells were lysed with RIPA buffer and 200 μg cell lysates were used for the immunoblot assay. To perform the immunoblot assay, OLA1 was detected using rabbit anti-OLA1 antibodies (Abcam Inc., Cambridge, UK) and GAPDH (internal loading control) using rabbit anti-GAPDH (Genetex Inc., Irvine, CA, USA), and they were visualized by chemiluminescence (Genetex Inc., Irvine, CA, USA) and imaged with a ChemiDoc-It^® 815 Imager (Analytik Jena, An Endress + Hauser Company, Jena, Germany) after incubation with HRP-conjugated secondary antibodies (Genetex Inc., Irvine, CA, USA).

Small molecule inhibitors are as follows: everolimus, dasatinib, duvelisib, alisertib, ibrutinib, idelalisib, sorafenib, metformin, and entospletinib (Selleckchem, Houston, TX; further information in Additional file 1: Table S1). All chemicals were from Sigma-Aldrich (St. Louis, MD) unless otherwise noted. Primary antibodies are as follows: rabbit anti-ATF-4 (#11815), -ATF-6 (#65880) and –β-tubulin (#2128) and mouse-anti-DDIT3/CHOP (#2895) (Cell Signaling Technology, Danvers, MA), rabbit anti-GAPDH (#100118; GeneTex, Irvine, CA), mouse anti-HSP70 (#648001; BioLegend, San Diego, CA), goat anti-Lamin-B (#SC-6217; Santa Cruz Biotechnology, Dallas, TX). Secondary antibodies are as follows: HRP-conjugated goat anti-rabbit (#111-035), goat anti-mouse (#115-035), and donkey anti-goat (#705-035) IgG antibodies (Jackson Immunoresearch, West Grove, PA).

Cells were washed twice with ice-cold PBS [(in mM) 137 NaCl, 2.7 KCl, 4.3 Na₂HPO₄. 2H₂O, 1.4 KH₂PO₄, pH 7.3] supplemented with 2 mM EDTA, and resuspended in a lysis buffer [(in mM) 150 NaCl, 5 EDTA, 50 Tris-HCl pH7.6, 1% Triton X-100) containing a protease inhibitor cocktail (Roche Applied Science). After adding the Laemmli sample buffer to the lysates, samples were sonicated on ice and heated at 70 °C for 5 min. Samples were then separated by 15% SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and detected using rabbit anti-GAPDH (1:5000; GeneTex), rabbit anti-Kv1.1 (1:2500; Upstate), or mouse anti-Myc (clone 9E10) antibodies. Blots were then exposed to horseradish peroxidase-conjugated anti-mouse/rabbit IgG (1:5000; Jackson ImmunoResearch), and revealed by an enhanced chemiluminescence detection system (Thermo Scientific). Results shown are representative of at least three independent experiments. Densitometric scans of immunoblots were quantified by using ImageJ (National Institute of Health).

UAS-Tau^WT and UAS-Tau^R406W transgenic flies, as previously described in Wittmann et al. 2001, were crossed with the pan-neuronal expression driver elav-GAL4 to generate experimental animals with the genotype elav-GAL4/+;UAS-Tau^WT/+ or elav-GAL4/Y;UAS-Tau^WT/+ (elav > Tau^WT) and elav-GAL4/+;UAS-Tau^R406W/+ or elav-GAL4/Y;UAS-Tau^R406W/+ (elav > Tau^R406W), respectively. These flies express the human Tau 0N4R isoform (383 amino acids). For control animals, we used the genotypes: elav-GAL4/+ and elav-GAL4/Y. All flies were raised on standard molasses-based Drosophila media at 25 °C with ambient light conditions, and aged to 1-, 10-, or 20-days following eclosion. We confirmed expression of Tau at similar levels in elav > Tau^WT and elav > Tau^R406W flies using western blot analysis, as previously described [31 (link)] using the following antibodies: rabbit anti-Tau (1:5000, Dako); rabbit anti-GAPDH (1:5000, GeneTex) and HRP-conjugated anti-rabbit (1:10000, Santa Cruz).