Mouse anti rabbit

Protein concentrations from total lysate supernatants were calculated (Pierce 660 nm protein assay reagent; Thermo Scientific, catalog no.: 22660), and samples were diluted to the same concentration in LDS sample buffer (National Diagnostics; catalog no.: EC-887) and heated in a 37 °C water bath for 15 min. Total lysate (5–10 μg total protein; typically one tenth of the 15% total lysate supernatant fraction) and IP samples (20 μl; 40% of eluent) were subject to SDS polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and incubated with 5% dry nonfat milk (Boston Bioproducts; catalog no.: P-1400) in PBS containing 0.1% Tween-20 (PBST). Membranes were then incubated with primary antibody in 1% milk in PBST as indicated in the text: mouse anti-GFP (Roche; catalog no.: 11814460001), rabbit anti-myc tag (Cell Signaling Technology; catalog no.: 2278), and rabbit anti-mCherry (ProteinTech; catalog no.: 26765-1-AP). Followed by washing in PBST, membranes were incubated with secondary antibodies conjugated to horseradish peroxidase in 1% milk in PBST: mouse anti-rabbit (Jackson ImmunoResearch; catalog no.: 211-032-171) and goat antimouse (Jackson ImmunoResearch; catalog no.: 115-035-174). Protein bands were visualized digitally following application of ECL reagent with the KwikQuant Imager (Kindle Biosciences; catalog no.: D1001).

Primary antibodies used in this study were as follows: anti-BRAT1 (IF 1:500, WB 1:50,000; Abcam, ab181855), anti-INTS11 (WB 1:1000; Novus Biologicals, NB100-60638), anti-INTS11 (IF 1:500; Novus Biologicals, NBP3-03680), anti-INTS9 (WB 1:1000; Cell Signalling, 13945), anti-INTS4 (WB 1:1000; Abcam, ab75253), anti-INTS3 (WB 1:1000; Bethyl, A302-050A), anti-INTS1 (WB 1:1000; Bethyl, A300-361A), anti-β-actin (WB 1:5000; Protein Tech, 66009), anti-α-tubulin (WB 1:8000; Abcam, ab6160), anti-Lamin B (WB 1:500; Santa Cruz, sc-6216), anti-Coilin (IF 1:250, WB 1:1000; Santa Cruz, sc-32860), anti-B23 (IF 1:250; Santa Cruz, sc-271737) and anti-FLAG (IF 1:250, WB 1:500; Sigma, F1804). Secondary antibodies employed for western blotting were HRP-conjugated goat anti-rabbit (1:10,000; Bio-Rad, 170-6515), goat anti-mouse (1:10,000; Bio-Rad, 170-6516), rabbit anti-rat (1:10,000; Abcam, ab6734), for western blotting after immunoprecipitation HRP-conjugated light chain specific mouse anti-rabbit (1:10,000; Jackson Immunoresearch, 211-032-171) and for indirect immunofluorescence were goat anti-rabbit Alexa 488 (1:10,000; Invitrogen, A-11008) and donkey anti-mouse Alexa 647 (1:10,000; Invitrogen, A-31571).

Tissue were homogenized in lysis buffer containing 100 mM Tris–HCl pH 7.5, 2 mM EDTA, 2 mM EGTA, 150 mM NaCl, 1% Triton X-100, cOmplete inhibitor cocktail (Roche) and PhosSTOP (Roche). Protein concentration was determined by Bradford assay. Equal amounts of protein were separated by SDS-PAGE and transferred onto nitrocellulose membranes (GE Healthcare). The nitrocellulose membranes were blocked with 5% BSA in TBST (TBS containing 0.1% Tween20) and incubated overnight in primary antibody diluted in TBST containing 5% BSA. Primary antibodies used were RICTOR (1:1000; Cell signaling; Cat#2140), AKT (1:1000; Cell signaling, Cat#4685), AKT-pS473 (1:1000; Cell signaling, Cat#9271), CALNEXIN (1:1000, Enzo, Cat#ADI-SPA-860-F), GAP43 (1:1000, Cell signaling, Cat#8945), GAP43-pS41 (1:1000, R&D Systems, Cat#PPS006), Tyrosine hydroxylase (1:500, Millipore, Cat#AB1542), HSL (1:2000, Cell signaling, Cat#4107), HSL-pS660 (1:1000, Cell signaling, Cat#4126), HSL-pS563 (1:1000, Cell signaling, Cat#4139). The primary antibody was washed several times with TBST and then incubated in secondary antibody in TBST containing 5% milk powder (w/v). Secondary antibodies used were mouse anti-rabbit (1:10′000, Jackson, 211-032-171) and rabbit anti-sheep (1:10′000, Invitrogen, 81-8620).

Samples were diluted to the same concentration in SDS sample buffer and heated in a 37 °C water bath for 15 min. Total lysate utilized 10–15 μg of total protein and IP utilized 20 μL from the preparation detailed above. Samples were resolved by SDS polyacrylamide gel electrophoresis, transferred to PVDF membranes, and incubated with 5% dry non-fat milk (LabScientific #M0841; Danvers, MA, USA) in PBS containing 0.1% Tween-20 (PBST). Membranes were incubated with primary antibodies in 1% milk in PBST: mouse anti-GFP (Santa Cruz; #sc-9996), rabbit anti-GFP (ThermoFisher; #A6455), and rabbit anti-myc-tag (Cell Signaling Technology #2278; Danvers, MA, USA). Following washing in PBST, membranes were then incubated with secondary antibodies conjugated to horseradish peroxidase in 1% milk in PBST: mouse anti-rabbit (Jackson ImmunoResearch #211-032-171; West Grove, PA, USA) and goat anti-mouse (Jackson ImmunoResearch #115-035-174). Protein bands were visualized digitally with the KwikQuant Imager (Kindle Biosciences #D1001; Greenwich, CT, USA) following application of ECL reagent. The predicted mass for each KCTD, based on UniProt ID, is listed in Supplementary Table S1.

The immuno-slot-blot technique was performed as previously described [52 (link)]. Briefly, after alkaline DNA denaturation (10 min 56°C, followed by 3N of NaOH), DNA was blotted on positively charged nitrocellulose membranes (Bio-Dot^® SF Microfiltration Apparatus), and DNA was heat fixed to the membrane (80°C, 3 h). Membranes were then blocked with 5% nonfat dry milk and hybridized with a mouse anti-CPD monoclonal antibody (Cosmo Bio Co., clone TDM-2) diluted 1:5 000 in 1% milk + 0.05% tween. The secondary HRP-conjugated antibody (Rabbit anti-mouse) (Jackson ImmunoResearch) was diluted 1:5 000 in 1% milk + 0.05% tween. A mouse anti-ssDNA monoclonal antibody (EMD Millipore, clone 16–19) diluted 1:1 000 was used to detect DNA. Membranes analysis and quantification were performed with C-DiGit^® Blot Scanner (LI-COR Biosciences).
NHDF from 3 different cell strains were used for this experiment, and the slot blot was performed at least twice for each NHDF strain cells. P-values were derived from the two-tailed heteroscedastic Student’s t-test.