Imagequant las 4000

Proteins isolated from the three HEK cell lines were prepared as previously described (Campiglio and Flucher, 2017 (link)). Briefly, cells plated in 100-mm dishes were trypsinized after 48 h in culture. Cells were lysed in radioimmunoprecipitation assay buffer with a pestle and left on ice for 30 min. The lysates were then centrifuged for 10 min. The protein concentration was determined using a BCA assay (cat. #23250; Pierce). 20 µg of protein samples were loaded on a NuPage gel (4–12% polyacrylamide, cat. #NP0321; Invitrogen) and separated by SDS-PAGE at 160 V. The protein samples were then transferred to a PVDF membrane at 25 V and 100 mA for 3 h at 4°C with a semidry blotting system (Roth). The membrane was then cut and incubated with rabbit anti-STAC3 (1:2,000; cat. #20392-1; Proteintech; RRID:AB_10693618) or mouse anti-GAPDH (1:100,000; cat. #sc-32233, Santa Cruz Biotechnology; RRID:AB_627679) antibodies overnight at 4°C and then with HRP-conjugated secondary antibody (1:5,000; Pierce) for 1 h at room temperature. The chemiluminescent signal was developed with ECL Supersignal WestPico kit (cat. #34579; Thermo Fisher Scientific) and detected with ImageQuant LAS 4000.

DIV 9–10 GLTs expressing STAC1-GFP, STAC2-GFP, STAC2-GFP and STAC3-NAM-GFP with or without Ca_V1.1 were scraped in RIPA buffer (50 mM TRIS-HCl, pH 8; 150 mM NaCl₂; 10 mM NaF; 0.5 mM EDTA; 0.10% SDS; 10% glycerol; 1% igepal; 1x Protease Inhibitor Complete cocktail (Roche)). The lysates were then purified by centrifugation (4,000 g, 10 min, 4 °C). Protein concentrations were determined using a BCA assay (Thermo Scientific) according to manufacturer instructions. Twenty micrograms of protein were separated by SDS-PAGE (4–12%) at 196 V and 40 mA for 60 min and transferred to a PVDF membrane at 25 V and 100 mA for 3 h at 4 °C with a semidry-blotting system (Roth). The blot was incubated with rabbit anti-GFP (1:10,000; Invitrogen) and mouse anti-GAPDH (1:100,000; Santa Cruz Biotechnology) antibodies overnight at 4 °C and successively with HRP-conjugated secondary antibody (1:5000; Pierce) for 1 h at room temperature. The chemiluminescent signal was detected with ECL Supersignal West Pico kit (Thermo Scientific) and visualized with ImageQuant LAS 4000.

Freshly isolated primary microglia were lysed on ice in cold RIPA lysis buffer containing: 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% IGEPAL CA-630 and 0.50% Na⁺ deoxycholate supplemented with protease inhibitor (Roche, Basel, Switzerland). Proteins were collected from the supernatant upon centrifugation and concentrations were determined using the bicinchoninic acid protein (BCA) assay kit (#23225, Thermo Fisher Scientific). Equal protein amounts (diluted in Laemmli buffer with 5% β-mercaptoethanol and heated for 4 min, at 95 °C) were used for SDS-PAGE (12% SDS-PAGE gel). Gels were transferred to a polyvinylidene fluoride (PVDF) membrane and blocked for 2 h with blocking buffer (Tris-buffered saline-0.1% Tween-20 containing 5% skimmed milk (Carl Roth, Karlsruhe, Germany)). Rabbit anti-p27 antibody (1:500, 25614-1-AP, Proteintech) was subsequently incubated overnight, at 4 °C, followed by washing steps and incubation with a horseradish peroxidase-conjugated secondary antibody (#G-21040, Invitrogen, Merelbeke, Belgium) for 1 h. All antibodies were diluted in blocking buffer, and incubations were at RT unless stated otherwise. Proteins were visualized using the enhanced chemiluminescence system (#32106, Pierce^TM ECL Western Blotting substrate, Thermo Fisher Scientific) and ImageQuant LAS 4000. Homogenous loading was checked using α-tubulin (#T9026, Sigma-Aldrich) staining.

PIP-2 concentrations of 3 µM, 6 µM, and 12 µM were used to treat A549 cells for 24 h. Using chilled RIPA cell lysis buffer, treated and untreated cells were lysed. The lysate of the cells was centrifuged for 15 min at 13,000 rpm at 4 °C. The BRADFORD method was used to quantify proteins in the cell lysate. A 70 g protein sample was isolated on a 12% SDS PAGE gel with a verticle separator. In a transfer buffer containing 25 mM Tris, 190 mM glycine, and 20% methanol, the gel was transferred onto a PVDF blot. Anti-c-myc (diluted to 1:1000 in TBST buffer) and anti-GAPDH (endogenous loading control diluted to 1:2000 in TBST buffer) antibodies were then incubated overnight on the blot membrane (from Thermo Fisher Scientific). The blot was then incubated for 2 h at 4 °C with an anti-mouse secondary antibody (Thermo Fisher) linked to Horseradish peroxidase diluted to 1:10,000 in TBST, and the image was analyzed using chemiluminescence in Image Quant LAS 4000. (GE Healthcare). Image J was used to examine relative band intensities.