Chameleon duo pre stained protein ladder

To analyze the point mutation case, 1016–15, (nominal concentrations of 8 μg and 16 μg total protein) was determined by bicinchoninic acid (BCA) assay (Pierce; Rockford, IL; cat. no. 23225) for each sample and was run on a Criterion™ TGX Any kD™ Precast Gel (BioRad; Hercules, CA; cat. no. 567–1125) alongside 5 μl of Chameleon Duo Pre-stained Protein Ladder (LI-COR; Lincoln, NE; cat. no. 928–60000) (marker) for 30 min at 25 mA and then 1.5 h at 150 V. Proteins were transferred to a nitrocellulose membrane at 30 V overnight at 4°C. The membrane was blocked in 1:1 PBS:Odyssey® Blocking Buffer (LI-COR; 927–40000) for 1 h at room temperature. Mouse anti-FMRP (Millipore; Burlington, MA; cat. no. MAB2160; Lot no. 2548166) primary antibody in blocking buffer, supplemented with 0.1% Tween-20, was applied to the membrane at 1:5,000 overnight at 4°C. Subsequently, IRDye® 800CW Goat anti-Mouse (LI-COR; cat. no. 926–32210; Lot no. C50316-03) secondary antibody in blocking buffer, supplemented with 0.1% Tween-20, was applied to the membrane at 1:20,000 for 1.5 h at room temperature. Membrane was visualized on the LI-COR Odyssey Infrared Imager.

Whole cell extracts for western blotting were obtained by applying RIPA buffer (Sigma-Aldrich, #R0278) supplemented with protease inhibitor (Cell Signaling Technology, #5872S) onto a cell monolayer and incubating on ice for 5 min. Following centrifugation (>16,000 × g) for 10 min), protein concentrations were quantitated using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, #23225). Equal amounts of protein were resolved and transferred according to manufacturer specifications, followed by overnight incubation at 4 °C with the primary antibodies listed in Supplementary Table 5. Chameleon Duo Prestained Protein Ladder (Licor, #928-60000) or PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, #26616) were used as a molecular weight marker. Densitometry analysis of each blot was quantitated using ImageJ. Uncropped immunoblots are provided in Source Data.

Cells were collected after treatment and lysed in a buffer of 50 nM Tris-HCl, pH 8, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.5% Triton X-100 and 1% SDS and freshly supplemented with protease inhibitors (Roche, 11873580001). Then, 15 μg whole lysates per lane and a Chameleon Duo pre-stained protein ladder (LI-COR, 928-60000) were resolved on 4–12% Bis-Tris gels (NuPAGE Invitrogen, NP0321BOX) and transferred to Amersham Protran 0.2-μm nitrocellulose membranes (GE Healthcare, 10600001). Blots were blocked with a LI-COR blocking buffer and incubated with the primary antibodies, namely anti-FTH1 (3998S, Cell Signaling), anti-β-actin (A5441, Sigma), anti-CD71/TfR (Cell Signaling, D7S5Z, 1:1,000 dilution), anti-ZIP14 (PA5-21077, Thermo Fisher, 1:1,000 dilution) and anti-GAPDH (ab9485, Abcam, 1:2,000 dilution) at 4 °C overnight and subsequently incubated with the corresponding secondary anti-IgG antibodies, anti-rabbit IRDye 680 CW (1:15,000 dilution, LI-COR, 926-68071), anti-rabbit IRDye 800 CW (1:15,000 dilution, LI-COR, 926-32211) and anti-goat IRDye 680 CW (1:10,000 dilution, LI-COR, 926-68074) for 1 h at room temperature. Blots were analyzed with an Odyssey Fc imaging system (LI-COR). For densitometry, OD of the protein of interest relative to the housekeeping gene was normalized to the averages obtained in control samples.

Confluent HFFs cultured in a six-well plate were treated with indicated concentrations of GSK-J4 or UNC1999 in 10% FBS DMEM for 4 days, with a media change including fresh inhibitor on day 2. Untreated cells were cultured in parallel. Histones were isolated from inhibitor-treated or untreated cells using the histone extraction kit (Active Motif, 40028), and western blots were performed. Histone extracts were combined with Li-cor 4X Protein Loading Buffer (928-40004) and resolved on Bio-Rad Mini-PROTEAN TGX 4-20% gel (4561094) in Boston BioProducts Tris-Glycine-SDS Running Buffer (BP-150), and transferred onto an Immobilon-FL PVDF membrane (IPFL00010) using Boston BioProducts Transfer Buffer (BP-190) made to 20% vol/vol methanol. Membranes were blocked in Odyssey Blocking Buffer (OBB, 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2% Fish gelatin, 1% ovalbumin) for a minimum of 1 hour at room temperature, washed with TBS-T (Research Products International T60075-4000.0 with 0.1% Tween 20, and incubated with primary antibody (diluted in OBB with 0.2% Tween 20) overnight at 4°C. Li-cor secondary antibodies (925-32211, 925-68070) were diluted in OBB (with 0.2% Tween 20, 0.02% SDS) and blots imaged on a Li-Cor Odyssey CLX-1374. Band intensity quantification was performed using Li-Cor Image Studio v5.2. Li-Cor Chameleon Duo Pre-Stained Protein Ladder (928-60000) was used.

Proteins and cell lysates were denatured by heating to 95°C for 5 min in LDS Sample Buffer (Thermo, NP0008) supplemented with 1X reducing agent (Thermo, NP0009), resolved using 4–12% Bis-Tris Protein Gels (Thermo, NP0329BOX) run in MES SDS Running Buffer (Thermo, NP0002), and transferred to nitrocellulose membranes (Thermo, IB23002) using the iBlot 2 (Thermo). Membranes were blocked with Odyssey Blocking Buffer in TBS (LI-COR, 927–50000) for 2 h at RT and incubated with primary antibodies in Odyssey Blocking Buffer + 0.2% Tween-20 (Sigma, P9416) overnight at 4°C. Membranes were washed three times with 1X TBST (G-Biosciences, R042) and incubated with secondary antibodies for 2 h at RT. After washing three times with 1X TBST, blots were imaged with the LI-COR Odyssey (LI-COR Biosciences, Lincoln, NE). Antibodies and reagents: GAPDH (Thermo, AM4300, 1:5000, 0.25 μg/mL), goat anti-rabbit (LI-COR, 926–32211, 1:15000), goat anti-mouse (LI-COR, 926–68020, 1:15000), Chameleon Duo Pre-Stained Protein Ladder (LI-COR, 928–6000).