Donkey anti rabbit 800cw

Two days prior to treatment, ~ 4 × 10⁵ MCF‐7 cells were plated on a 6‐cm dish. Cells were treated with either 400 ng/ml neocarzinostatin (Sigma, N9162), 5 μM Nutlin‐3 (Sigma, N6287), 10 μM Nutlin‐3, or 15 μM Nutlin‐3 for 3 h. Cells were harvested by scraping and frozen in a dry ice‐ethanol bath. Cells were lysed in M‐PER Mammalian Protein Extraction Reagent (Thermo Fisher, 78501) according to manufacturer's protocol, and the concentration for each protein sample was determined by Bradford assay. Equivalent protein masses were separated by electrophoresis on 4–20% gradient gels (Bio‐Rad, 456‐1096) and blotted onto Immobilon‐P PVDF membranes (Millipore, IPVH00010). Membranes were incubated with the following primary antibodies: mouse monoclonal anti‐p53 DO‐1 (Santa Cruz Biotechnology, sc‐126) and rabbit monoclonal anti‐β‐actin 13E5 (Cell Signaling Technology, 4970). Membranes were then incubated with the following secondary antibodies: donkey anti‐mouse 680RD (LI‐COR, 926‐68072) and donkey anti‐rabbit 800CW (LI‐COR, 926‐32213). Membranes were imaged with a LI‐COR Odyssey system (LI‐COR) and quantified using LI‐COR Image Studio v3.1 software. Following background subtraction, the band measurements were normalized to β‐actin measurements in the same lane.

To quantify protein depletion, Western blots were performed with cell lysates. Parallel prepared samples of the siRNA treated cells were lysed at the time of imaging in high salt radioimmunoprecipitation assay (HS RIPA) buffer. Lysates were vortexed for five minutes to shear DNA, heated to 93°C for two minutes, and centrifuged to pellet DNA. Protein concentrations were determined by Bradford assay. Equal amounts of protein (15 μg) for each sample were loaded into a NuPAGE 10% Bis-Tris gel (Gibco). Gels ran in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer at 100 V until the ladder bands began to separate, and then at 175 V until completion. Proteins were then transferred to a polyvinylidene difluoride (PVDF, Millipore) membrane at 16 V for one hour. Gels were stained with Coomassie to observe quality of protein loading. After transfer, the membrane was blocked for at least one hour at room temperature in 5% milk. Primary antibodies (anti-lamin A/C, Santa Cruz, sc-6215, dilution 1:2000; anti-CHMP7, Sigma, HPA036119, dilution 1:200; anti-actin, Santa Cruz, sc-1615 HRP, dilution 1:2000) were added and left on overnight at 4°C. Secondary antibodies (donkey anti-rabbit 800 cw and donkey anti-mouse 680 RD, Licor) were incubated for one hour at room temperature prior to imaging on a LI-COR Odyssey CLx.

All animal studies and breeding was approved by the University of Dundee ethical committee and performed under a UK Home Office approved project license. Snap frozen kidneys from CUL3^+/+ and CUL3^Δ403‐459 mice were sliced into quarters, and one of the quarters was homogenized using a TissueLyser LT ( https://www.qiagen.com/#85600) and protein lysates were extracted in RIPA lysis buffers containing protease and phosphatase inhibitors ( https://www.roche.co.uk/ #11836170001 & #04906845001). All steps were carried out at 4°C. Protein concentrations were determined with the Pierce^TM BCA protein assay ( https://www.thermofisher.com/#23225). 10 μg equivalent protein lysates were immunoblotted with the rabbit anti‐KCNJ1 antibody ( https://atlasantibodies.com/) at 2 μg/mL concentration in 5% (w/v) skimmed milk in TBS‐T. Secondary antibodies (donkey anti rabbit 800CW (926–32213 at 1:5000 dilution; www.licor.com) and goat anti mouse Alexa680 (A‐21058 at 1:5000 dilution; www. lifetechnologies.com) were incubated in TBS‐Tween for 1 h at room temperature in the dark, then washed 6X in TBS‐Tween. Membranes were imaged and integrated intensity values quantified (bands were normalized against β‐actin) using the LiCor odessey system ( www.licor.com) or using IMAGE J software ( http://imagej.net).

Cells were lysed in Cell Lysis buffer (20 mM triethanolamine (T1377; Sigma), 0.14 M NaCl, 0.1% Sodium deoxycholate (D6750; Sigma), 0.1% SDS, 0.1% Triton X-100) with Protease Inhibitor Cocktail (27368400; Roche), Phosphatase Inhibitor Cocktail (04906837001; Roche) and 10 mM N-Ethylmaleimide (NEM) on ice. BCA kit (23225; Thermo) was used to measure protein concentration. Equal amounts of total cell extracts were loaded on a NuPAGE gel (4–12%, NP0321; Thermo), and transferred to a Nitrocellulose Blotting Membrane (10600016; Life Sciences). The following primary antibodies were used: Morc3 (Rockland; 100-401-N96S 1:1000) and Tubulin (T6199; Sigma, 1:5000). Donkey anti-Rabbit 800CW (926-32213; Li-Cor, 1:5000), Goat anti-Rabbit 800CW (926-32211; Westburg, 1:5000), Donkey anti-mouse 680RD (926-68072; Li-Cor, 1:5000) were used as secondary antibodies. Membranes were analyzed on Odyssey (Westburg).

The Src inhibitor dasatinib (ChemieTEK, Indianapolis, IN) and the p300 inhibitor C646 (Sigma Aldrich, St. Louis, MO) were purchased from the designated sources. Antibodies used were as follows: Src (B-12), p300 (C20), GAPDH (6C5), β-Tubulin (C10), Grim19, (Santa Cruz Biotechnology, Santa Cruz, CA), Src (36D10), pY-416 Src (2101), HMGA2 (D1A7), SMYD3 (D2Q4V), Lamin A/C (#2032), Calnexin (C5C9), HDAC1 (10E2), Histone H3 (D1H2), p-TYR-100 (Cell Signaling Technology, Danvers, MA), pY418 Src (92633), p300 (507) (Novus Biologicals, Littleton, CO), HMGA2 (AF3184) (R&D Systems, Minneapolis, MN), and SMYD3 (ab16027) (Abcam, Cambridge, UK). Secondary antibodies used were Donkey-anti-Rabbit 800CW and Goat-anti-Mouse 680LT (Licor, Lincoln NE).

Samples were quantified by BCA assay (Pierce) and 25 µg of protein was analyzed per sample. Samples were separated on a 14% SDS-PAGE gel and transferred to nitrocellulose membrane (Bio-Rad). Membranes were blocked for 1 h at room temperature in 1% milk/TBS-T and then incubated in primary antibody overnight at 4°C. Primary antibodies were purchased from Cell Signaling (Danvers, MA): monoclonal rabbit anti-Stat1 antibody at 1:1,000 dilution, Cat. No. 14994S; polyclonal rabbit antiphospho-Stat1 (Ser727) antibody at 1:500 dilution, Cat. No. 9177S; monoclonal rabbit anti-Stat6 at 1:500, Cat. No. 5397S; and monoclonal rabbit anti-phospho-Stat6 (Tyr641) at 1:500, Cat. No. 56554S. The following day, samples were washed in Tris-buffered saline-Tween 20 (TBST), incubated with secondary antibodies (donkey anti-rabbit 800CW or donkey anti-mouse 680RD, 1:20,000, LI-COR) for 1 h at room temperature, washed with TBST, and imaged on an Odyssey Clx (LI-COR). Protein quantification was performed using ImageStudio Software (LI-COR).