Anti his tag

Standard kinase assays were performed at 30°C for 5-min incubation with 50 nM purified CaMKII proteins, 800 nM CaM, 1 mM CaCl₂, and 1 mM ATP in a reaction buffer [50 mM tris-HCl (pH 7.4), 150 mM KCl, and 10 mM MgCl₂]. To release CaM from CaMKII, 2 mM EGTA was added and incubated for 5 min at 30°C and an additional 10 min at 25°C to match the conditions for AFM observation (i.e., after the sample was loaded on the AFM apparatus, it typically takes 10 min before observation). For substrate phosphorylation, sfGFP-Syn2 was added at 1 μM and incubated for 7 min at 25°C. For CaMKII dephosphorylation by PP2A, phosphorylated CaMKII proteins were incubated with the indicated concentration of PP2A for 10 min at 25°C. The reactions were stopped by adding SDS sample buffer and then analyzed by Western blotting.
Western blotting was performed with the following antibodies: anti–phospho-CaMKII (Thr²⁸⁶) (D21E4; Cell Signaling Technology, USA), anti-pT305 (abx012403; Abbexa), anti–phospho-CaMKII (Thr³⁰⁶) (p1005-306; PhosphoSolutions, USA), anti–phospho-PKA substrate (RRXS*/T*) (#9624; Cell Signaling Technology), anti–His-tag (27E8; Cell Signaling Technology, USA), and HRP–anti-rabbit/mouse (Jackson Laboratory, USA).

WB and IFA were performed as described with antibodies, and the dilutions used were as follows: anti-Myc (Cell Signaling) 1:3000; anti-HA (Cell Signaling) 1:3000, anti-H3 (Abcam) 1:10000, anti-BIP 1:1000 (polyclonal), anti-H2A. Z (polyclonal–kindly provided by Dr. Nicolai Siegel), anti-His Tag 1:3000 (Cell Signaling); and anti-GAPDH 1:4000 (polyclonal) [89 (link)]. For IFA of myc-tagged parasites, after fixation with 4% paraformaldehyde and permeabilization with Triton X-100, an overnight incubation with anti-Myc (Cell Signaling) antibody 1:3000 in 1% PBS/BSA at 4°C followed by secondary antibody conjugated to Alexa Fluor 555 (Thermo Fisher) at 1:500 in PBS/BSA 1% was performed. When mentioned, IFA using anti-GP90 and anti-GP82 1:1000 (kindly provided by Nobuko Yoshida) was performed as described previously [90 (link)]. Slides were further mounted with Vectashield plus DAPI (Vector laboratories) and visualized at 100 X magnification in an inverted OLYMPUS microscope model IX81 with Z axis motorization. Images were acquired using OLYMPUS CELL R version software 3.2.

Total proteins were extracted from GC tissues or cells using RIPA lysis buffer containing protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). The lysates were mixed with SDS loading buffer, boiled for 8 min, resolved by SDS-PAGE, and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies: anti-LDHA (#3582S, Cell Signaling Technology), anti-HA (clone 3F10, #11867423001, Roche), anti-FLAG M2 (#F1804; Sigma), anti-His TAG (#12698S, Cell Signaling Technology), pan succinyl-lysine antibody (#PTM-401; PTM Bio, Hangzhou, China), anti-β-actin (#4970; Cell Signaling Technology), or anti-CPT1A antibody (#12252; Cell Signaling Technology).

The protein of cells was extracted for immunoblotting as described previously [18 (link),19 (link),20 (link)]. Briefly, the cells were harvested with lysis buffer containing protease inhibitor cocktail. The proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes for further immunoblotting with the primary antibodies, including anti-DYRK1B (1:1000, 5672, Cell Signaling Technology), anti-His tag (1:1000, 12698, Cell Signaling Technology), and anti-ACTB (1:2000, A5441, Sigma) at 4 °C overnight, followed by probing with secondary antibody and ECL reagent. The membrane was scanned and analyzed for protein expression as uncropped images in Figure S1 with a BioSpectrum^® Imaging System (UVP, Upland, CA, USA).

Cells were seeded on poly-d-lysine/laminin B coated cover glass and grown for 48 h in DMEM/F-12 (1:1) supplemented with GlutaMAX, 10% FBS, and 500 µg/mL P/S. For stably transfected cells, 300 µg/mL geneticin was added. After 48 h, the media was changed to differentiation media (Neurobasal media (#21103-49, Gibco) (Paisley Park, UK)) supplemented with 0.5 mM GlutaMAX (#35050-038, Gibco) (Paisley Park, UK), 1xB27 (#1704044, Gibco) (Paisley Park, UK), and 1% P/S (300 µg/mL geneticin for stabile transfectants). Ten µM retinoic acid (#R2625, Sigma) (Merck, Darmstadt, Germany) was added to the differentiation media for the first 5 days, followed by 5 days with 10 ng/mL BDNF (#450-02, PreproTech) (Thermo Scientific, Paisley Park, UK). Cells were fixed for 15 min in 4% formaldehyde and permeabilized in 0.25% Triton-X for 10 min. The proximity ligation assay (PLA) was conducted with the Duolink In Situ Red Starter Kit Mouse/Rabbit (#DUO92101, Sigma) (Merck, Darmstadt, Germany) according to manufacturer’s protocol. Cells were incubated with primary antibodies as indicated: anti-His-tag (Cell Signaling, #12698, 1:600) (Danvers, MA, USA), pan anti-PKC (Santa Cruz, #sc-17769, 1:1000) (Dallas, TX, USA), and anti-CDK5 (Santa Cruz, #sc-6247, 1:100) (Dallas, TX, USA). PLA signals were visualized using an Axio Observer microscope and Zen Pro software (Zeiss, Oberkochen, Germany).

SnMP, DNR hydrochloride, poly(lactic‐co‐glycolic acid) (PLGA, lactide: glycolide 50:50, 7000–17000 Da), and biotin‐FITC were purchased from Sigma Aldrich (St. Louis, MO, USA). 1,2‐Dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) and 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[biotinyl(polyethylene glycol)‐2000] (DSPE‐PEG₂₀₀₀‐Biotin), 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[amino(polyethylene glycol)‐2000] (DSPE‐PEG₂₀₀₀) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Anti‐human CD33, CD64 antibodies and anti‐mouse CD11b, CD45, CD206, Ly6c, Gr1, TNF‐α, IL12p70, Rat IgG1 Isotype antibodies were purchased from BD Biosciences (USA) (Table 1, Supporting Information). Anti‐mouse F4/80 antibody and Avidin‐FITC were purchased from Biolegend (San Diego, CA, USA). Anti‐human CCR2 antibody was obtained from R&D Systems (Minneapolis, MN, USA). Anti‐His‐Tag, anti‐human HO1 (P249), and β‐actin antibodies (13E5) were obtained from Cell Signaling Technology (Danvers MA, USA).