Ab5804

Micro-channels were coated with fibronectin (Sigma) or PLL(20)-g[3.5]-PEG(2) (SuSoS Chemical). For myosin light chain kinase inhibition, cells were incubated with different concentrations of ML7 from Calbiochem as indicated for 16 h. For Ca²⁺ experiments, Oregon Green BAPTA 1-AM, FuraRed, BAPTA (Invitrogen), and Thapsigargin from Calbiochem were used. For IP₃R inhibition, 5 μM xestospongin C from Calbiochem was used. For dendritic cell maturation, we incubate the cells 24 h with 100 ng/ml LPS (Sigma). For flow cytometry analysis, we used a homemade 24G2 anti-Fc Receptor antibodies, rabbit serum from Agro Bio as a control, and anti-CD11c (HL3 clone), anti-IAb^b (AF6-120.1 clone), and anti-CD86 (GL1 clone). For immunoblot, we used anti-IP₃R type 1 (Abcam ab5804), anti-IP₃R type 3 (610313 BD Transduction Laboratories), anti-phospho-myosin light chain (Rockland 600-401-416), and anti-actin (Millipore). For lentivirus production, HEK cells were transfected using GeneJuice (Novagen).

Western blotting analysis was performed as described previously[17 (link)] with minor modifications. SM samples were homogenized in RIPA buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) to extract protein. Samples were heated at 90°C for 5 min and centrifuged at 5000 ×g for 5 min. The supernatants were resolved on 10% (for large-conductance calcium-activated potassium channel [BKCa], SERCA2a and nuclear factor-kappa B [NF-κB]) or 8% (for IP3R1) SDS-polyacrylamide gels and 4–12% Bis-Tris Gels (Invitrogen, NY, CA, USA) (for phospholamban [PLB]). The resolved protein bands were transferred to PVDF membranes (Millipore, Bedford, MA, USA) at 300 mA for 1 h (BKCa, NF-κB), 300 mA for 1.5 h (SERCA2a), 400 mA for 1.5 h (IP3R1), or 400 mA for 1 h (PLB). The membranes were blocked with Tris-buffered saline-Tween 20 containing 5% nonfat dry milk (Sigma-Aldrich) at room temperature for 1 h and then incubated at 4°C overnight with anti-SERCA IgG (1:1000, ab2801, Abcam, Cambridge, UK), anti-BKCa IgG (1:1000, ab3587, Abcam), anti-NF-κB IgG (1:2000, 8242, Cell Signaling Technology, Inc.), anti-IP3R1 IgG (1:1000, ab5804, Abcam) or anti-PLB IgG (1:1000, ab2865, Abcam). The membranes were washed and then incubated with the appropriate secondary antibodies at room temperature. After washing, labeled bands were detected using enhanced chemiluminescence solutions 1 and 2 (1:1) (Millipore).

Primary antibodies for Western blot: SBDP (1:20; MAB1622, EMD Millipore) and PAD-tau (1:20; MABN417, EMD Millipore). Primary antibodies for IHC: calpain-1 (1:200; LS-B4768, LSBio), calpain-2 (1:300; LS-C337641, LSBio), GFAP (1:1000; AB5804, Abcam), iba-1 (1:400; AB5076, Abcam), p-tau Thr²³¹ (1:200; MN1040, Thermo Fisher Scientific), p-TDP-43 409/410 (1:400; 22309-1-AP, Proteintech), and NeuN (1:200; ab104224, Abcam). Secondary antibodies for IHC: Alexa Fluor 594 goat anti-rabbit immunoglobulin G (IgG) (1:400; A11037, Thermo Fisher Scientific), Alexa Fluor 594 goat anti-mouse IgG (1:400; A11005, Thermo Fisher Scientific), and Alexa Fluor 594 donkey anti-goat IgG (1:400; A11058, Thermo Fisher Scientific).