Complete mini protease inhibitor cocktail tablet

For cellular protein extraction, one million cells were lysed using RIPA buffer (50 mM Tris-HCl pH8, 150 mM NaCl, 1% Triton X100, 0.5% deoxycholate, 0.2% SDS and 0.2 mM EDTA) supplemented with an antiprotease pellet (cOmplete^TM Mini Protease Inhibitor Cocktail Tablets, Roche Diagnostics, Meylan, France). SDS-PAGE electrophoresis (10%) was subsequently performed (50 μg proteins/lane). Proteins from the gels were electrotransferred onto Hybond ECL membrane (Amersham-GE Healthcare, Velizy-Villacoublay, France). After blocking with 5% non-fat dry milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (“Tris buffer”) for 1h at room temperature, blots were washed three times with Tris buffer and probed 3h with specific primary antibody in 5 % non-fat dry milk in Tris buffer. The primary antibodies used against Orai1 (1:1000), STIM1 (1:1000), cyclin D1 (1:500) and tubulin (1:3000) were from Sigma-Aldrich, Saint Quentin Fallavier, France). The anti-tubulin antibody was used to verify that equal amounts were loaded in each lane. Detection was performed using the enhanced chemiluminescence reagent (ECL Western blotting detection reagents, Amersham-GE Healthcare). Quantification of the bands was performed with the ImageJ software.

Plating media was removed from microfluidic chambers and culture was rinsed with HBSS. Axons were harvested with RIPA buffer with protease (Complete^TM Mini Protease Inhibitor Cocktail tablets, Roche) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail, A.G. Scientific), with protein pooled from 2 to 4 chambers per treatment group. Denatured protein samples (20 μg) were electrophoresed into Bolt^®; 4–12% Bis-Tris gels (Invitrogen), transferred to PVDF membrane (Bio-Rad) and incubated overnight with primary antibodies acetylated tubulin (1:1,000 mouse, Sigma), tyrosinated tubulin (1:1,000 rabbit, Millipore), MAP1B (1:1,000 mouse, AbCam). The corresponding anti-rabbit or anti-mouse horseradish peroxidase conjugated secondary antibody (1:7,000, DAKO) was used as previously described (King et al., 2018 (link)). GAPDH (1:5,000 mouse, Millipore) was used as a loading control. Bands were visualized with enhanced chemiluminescence (ECL) solution-Luminata Forte Western horseradish peroxidase (HRP) substrate (Millipore) and images acquired with a Chemi-Smart 5000 Imaging System (Vilber Lourmat) equipped with Chemi-Capt 5000 software. Band intensity was measured as the integrated intensity using Fiji software. After standardizing to GAPDH, each value was calculated as a percentage of control samples (See Supplementary Figure 1).

Bio‐Plex Pro 23‐Plex mouse cytokine kit (Bio‐Rad) was used to measure muscle cytokine levels in powdered gastrocnemius muscle as per manufacturer's instructions with minor modifications (n =4 per condition). Briefly, muscle tissue was washed three times in wash buffer, and put in ice‐cold Bio‐Plex cell lysis buffer containing factors 1 and 2 from the tissue lysis kit, PMSF, and one complete mini protease inhibitor cocktail tablets (Roche Applied Science) per 50 mL of buffer. The tissue preparation was transferred to an ice‐cold tissue homogenizer and dounced 25 times on ice. The sample was transferred to a new tube and placed at −80°C for 1 h. This was followed by sonication (Tekmar Sonic Disrupter, Cincinnati, OH) of the tissue on ice for 20 sec for five times at 50% power. The extracts were collected by centrifugation at 3400g at 4°C for 30 min, and the samples were transferred to a new tube and aliquoted at −80°C until further processing. The samples were thawed only once at the time of processing. Standard curves were generated and cytokine measurements were normalized to total protein content of the sample and expressed as pg μL⁻¹μg⁻¹ protein.