A21057

1 × 10⁶ cells were lysed directly into 2.5x Laemmli sample buffer, separated by 8% SDS-PAGE, and transferred to PVDF-FL membranes (Millipore). Membranes were blocked in 5% milk in PBS and incubated with primary antibody diluted in 5% milk in PBS supplemented with 0.1% Tween20. Secondary antibodies were diluted in 5% milk in PBS supplemented with 0.1% Tween20 and 0.01% SDS. Membranes were imaged with a Licor Odyssey instrument. Primary antibodies used in these experiments were rabbit anti-Cas9 (Abcam ab204448) and mouse anti-HSP90 (BD Transduction Laboratories 610418). Secondary antibodies used were goat anti-rabbit IRdye 800CW (Licor 827-08365) and goat anti-mouse Alexa Fluor 680 (Molecular Probes A-21057). Relevant regions of each immunoblot are shown in Figs 1 and 3, and full images are provided in the supplement.

All cells (5 × 10³ cells/well) were seeded in 96-well plates and cultured overnight. The media was replaced with drug-supplemented (e.g., encapsulation-DOX, covalent conjugation-DOX and PEG coat-DOX) media at 100 μl/well and incubated for the indicated time. Cells were fixed with 4% paraformaldehyde in medium overnight at 4 °C. Permeabilization was performed in PBS with 0.1% Triton X-100 for 15 min at RT. After a blocking period of 2 hrs with 1% BSA in PBS, cells were incubated with RAB2 (polyclonal, ab131568, Abcam), RAB7 (polyclonal, ab50533, Abcam), RAB9 (polyclonal, ab179815, Abcam) and RAB11 (polyclonal, ab3612, Abcam) antibodies overnight at 4 °C in the absence of light. After washing twice times in PBS, cells were incubated with Alexa Fluor 405 Goat Anti-Rabbit IgG (H + L) and Alexa Fluor 405 Goat Anti-Mouse IgG (H + L) (1:200, A11008, A21057, Molecular Probes) for 2 hrs at RT. Cells were washed twice times in PBS and fluorescence was measured with a microplate reader (VICTOR X3, PerkinElmer, Waltham, MA, USA) at 405 nm (excitation) and 460 nm (emission).

Western blotting was performed as described previously (66 (link)). Briefly, protein preparations were mixed with 5× Laemmli loading buffer and heated to 95°C for 10 minutes. Equal amounts of protein were subjected to electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, IPFL00010). The membranes were incubated with antibodies against Hsp47 (1:000, Novus Biologicals, M16.10A1), Gapdh (1:10000, Fitzgerald, 10R-G109a), or α-tubulin (1:1000, Santa Cruz, sc-8035). PVDF membranes were then incubated with the appropriate Alexa Fluor–conjugated secondary antibodies (Thermo Fisher Scientific, A21057, Goat anti-Mouse IgG Cross-Adsorbed Secondary Antibody, Alexa Fluor 680, 1:5,000; Thermo Fisher Scientific, A11367, Goat anti-Rabbit IgG Cross-Adsorbed Secondary Antibody, Alexa Fluor 790, 1:5000) and then visualized by using an Odyssey CLx imaging system (LI-COR Biosciences, 9140).

A standard procedure was carried out as described previously [36 (link)]. Briefly, coronal sections (8 μm) were prepared at the level of the dorsal hippocampus (1.90–2.50 mm posterior to the bregma). Paraffin sections were dried, washed, permeabilized, blocked in 5% goat serum, and incubated overnight at 4 °C with antibodies against goat anti-SerpinA3N (RD; AF4709), mouse anti-NeuN (Millipore; MAB377), rabbit anti-Olig2 (Abcam; ab109186), rabbit anti-GFAP (Abcam; ab4648), rabbit anti-IBA1 (Huabio; ET1705-78), rabbit anti-phospho-RYR2 (Ser2808) (Biorbyt; orb1093816), and mouse anti-RYR2 (Thermo Fisher Scientific, MA3-916), followed by an Alexa Fluor 680-, 594-, or 488-conjugated secondary antibody (Thermo Fisher Scientific, A-21057, A-11058, A-11001, A-11008) for 1 h at room temperature. Finally, the cells were incubated with mounting medium (with DAPI) (Cat#S2110, Solarbio, Beijing, China) at room temperature for 15 min. Histological images were captured using a Pannoramic MIDI scanner (3D Histech, Hungary). The experiment was repeated in triplicate. ImageJ software was used to manually count positive cells in the same size field of view.

Protein lysates from cell lines were prepared by resuspension in RIPA lysis buffer (ThermoFisher, 89900) followed by freezing at −20°C. Protein lysates from tumors were prepared by homogenization of flash frozen tumor tumors in RIPA buffer using Dounce homogenizers. Genomic DNA was sheared by centrifuging lysate in a QIAshredder column (Qiagen #79654). Protein assay was performed using the Lowry method (Bio-rad, 5000111). Electrophoresis was run using a bis-tris gel (Thermo Fisher, NW04122BOX), protein was transferred to a PVDF membrane (ThermoFisher, IB401001) using the iBlot2 transfer system (ThermoFisher, IB21001). Membrane was blocked using fluorescent blocking buffer (ThermoFisher, 37565). Membranes were incubated in anti-DNMT1 1/1,000 (Novus Biologicals, 60B1220.1), anti-survivin 1/200 (Abcam, ab469), anti-IRF5 1/500 (Cusabio, CSB-PA011820LA01HU), or anti-GAPDH 1/1,000 (Santa Cruz Biotech, sc-32233) overnight followed by incubation in fluorescent secondary antibody (ThermoFisher, A21057 or A11369) for 1 h at room temperature. Signal was visualized using the iBright imaging system (ThermoFisher, FL1500).

Mouse antibody to α‐Tubulin‐FITC (1:400, F2168) was purchased from Sigma (USA). Mouse antibodies to γ‐Tubulin (1:200, ab11316) and TPM3 (1:100, ab113692) were purchased from Abcam (USA). Mouse antibody to FLAG (1:2000, M20008L) was purchased from Abmart (Shanghai, China). Rabbit antibody to MYC (1:1000, BE2011) was purchased from EASYBIO (Beijing, China). Rabbit antibodies to β‐Actin (1:2000, AC026) and β‐Tubulin (1:1000, AC008) were purchased from ABclonal (Wuhan, China). Rabbit antibodies to RNF20 (1: 1000, 21625‐1‐AP) and TPM3 (1:1000, 10737‐1‐AP) were purchased from Proteintech Group (USA). Rabbit antibodies to H2Bub (1: 1000, 5546s), H2B (1: 1000, 12364S) and H3 (1: 1000, 4499S) were purchased from Cell Signaling Technology (USA). Mouse antibody to HEC1 (1: 100, sc‐515510) was purchased from Santa Cruz Biotechnology (USA). Rabbit antibodies to RBBP7 (1:100, bs8620) and UBASH3B (1:100, bs8741) were purchased from Bioworld (Beijing, China). Human antibody to ACA (1: 200, 15–234) was purchased from Antibodiesinc (USA). Goat anti‐rabbit FITC (1:200, ZF‐0311) and goat anti‐mouse TRITC (1:200, ZB‐2305) were purchased from ZSGB‐BIO (Beijing, China). Alexa Fluor 680‐conjugated goat anti‐mouse (1:10000, A21057) and Alexa Fluor 800‐conjugated goat anti‐rabbit (1:10000, A21109) were purchased from Invitrogen (USA).