Blocking buffer

The LKR-13 K-RasG12D lung adenocarcinoma cell line was obtained from J. Kurie (MD Anderson). The reagents obtained, used and cells were cultured in RPMI 1640 as previously described [3 (link)]. Immunofluorescence (IF) Ab for perforin and PD-L1 were from CST, granzyme B conjugated to Alexa 647 and Pan-CK were from Abcam, CD4 and CD8 conjugated to Alexa 488 were from Novus Biological. IF buffers were from DAKO and blocking buffer was from Thermo Fisher. For in vivo experiments, anti-PD-1 (BE0146), anti-CD4 (L3T4) anti-CD8 (YTS169.4), and anti- CXCL9 (MIG-2F5.5) were from BioXCell as previously described [3 (link)]. Goat anti-mouse CXCL10 was given by Dr. Robert Strieter. Isotype control Ab was from Sigma. Tissue digestion buffer was from Miltenyi and used according to manufacturer’s instructions. T cell purification columns were from R&D. RNA isolation kit was from Qiagen.

The colon tissue was lysed by the same procedure as described for the ELISA assay of spleen tissues. After quantification, 20 μg of protein samples were loaded onto 8%, 10%, and 12% SDS-PAGE gels for analysis of the expression of occludin (ab216327), claudin1 (ab15098), claudin2 (ab53032), claudin4 (ab15104), Zonula occludens-1 (ZO-1) (ab96587), CCL-20 (ab9829), HIF-1α (ab2185), and β-actin (ab16039) as loading control (Abcam, Cambridge, MA, USA). After transferring onto PVDF membranes (Merck Millipore Ltd., Burlington, MA, USA), and soaking in the blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min, the membranes were incubated with primary antibodies at 4 °C overnight. Then, the membrane was washed thrice with TBS-Tween buffer, and incubated in horseradish peroxidase (HRP)-labeled rabbit secondary antibodies (Abcam, Cambridge, MA, USA) for an hour. Finally, the membranes were immersed in western lighting ECL pro reagent (Perkin-Elmer, Waltham, MA, USA), and analyzed using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). The quantification of protein expression was performed in ImageJ (n = 8–10) [82 (link)].

As the mutation specifically affects the fast skeletal myosin heavy chain type IIx and as fibre typing for individual cells occurred after the mechanical experiments, we only kept the biophysical data from fibres expressing this specific isoform, i.e., the fast skeletal myosin heavy chain type IIx. Briefly, after the Mant-ATP chase experiments or contractile measurements, individual fibres were stained with an anti-myosin fast/type IIx antibody (IgM isoform, clone 6H1, dilution: 1:50, gift from Dr. Joseph Hoh—University of Sydney, Sydney, Australia) [24 (link)]. Myofibres were then washed in PBS/0.025% Tween-20, and incubated with a secondary antibody conjugated to Alexa 594 in a blocking buffer (from ThermoScientific, Waltham, MA, USA, dilution 1:500). After washing, muscle fibres were mounted in Fluoromount and images taken with a confocal microscope (Zeiss Axiovert 200, objective ×40) equipped with a CARV II confocal imager (BD Biosciences, Franklin Lakes, NJ, USA) [25 (link)]. The Supplementary Figure S1 depicts the pH 4.4 ATPase activity of fibres within the muscles of a control horse, a heterozygote, and an animal homozygous for the MYH1 mutation.

Briefly, RNA for Northern blotting (NB) was extracted using TRIzol (Life Technologies) according to the manufacturer’s instructions. RNA (20 μg) was mixed with 2×RNA loading buffer (TAKARA) and EB, denatured at 65°C for 15 minutes and then run on a 1.2% agarose/formaldehyde gel and transferred by capillary action onto a nitrocellulose membrane (Millipore). The nitrocellulose membrane was prehybridized with ExpressHyb solution (Clontech) at 42°C for 2 hours. Probes were produced using a North2South Biotin Random Prime DNA Labeling Kit (Thermo Scientific). Primers used for the production of probes are shown in Table 1. The membrane was hybridized at 42°C overnight with fresh solution containing the corresponding probe and then washed twice at room temperature for 30 min with wash solution 3 (2×SSC, 0.1% SDS) and once at 42°C for 30 min with wash solution 2 (0.1×SSC, 0.1% SDS). The membrane was then blocked with blocking buffer (catalogue #89880A; Thermo Scientific) at room temperature for 30 min. Finally, the membrane was incubated with IRDye 800-conjugated streptavidin diluted in TBST (1:2500) and imaged on an Odyssey CLx infrared imaging system (Li-COR Biosciences).

Immunoblot was performed to confirm the intracellular signaling pathway through the BDNF–TrkB pathway. To induce differentiation of SH-SY5Y cells (Korea Cell Line Bank), 10 μM of retinoic acid (Sigma-Aldrich, Saint Louis, MO, USA) was used. Recombinant BDNF protein (Peprotech, Rocky Hill, NJ, USA) was treated as positive control and supernatant of BDNF-eMSCs, together or alone with recombinant human TrkB-hyFc (Sino Biological, Beijing, China), were incubated with differentiated SH-SY5Y cells for 1 h at the 37 °C, 5% CO₂ incubator. BDNF-eMSCs were harvested and lysed with RIPA buffer (Thermo Fisher Scientific). Lysate proteins were distinguished using SDS-PAGE, transferred to nitrocellulose membranes (Thermo Fisher Scientific), and incubated with blocking buffer (Thermo Fisher Scientific) for 30 minutes. Immunoblots were performed using primary antibodies against TrkB, PLC-γ, AKT, ERK, Phospho-TrkB (Tyr706/Tyr707), Phospho-PLC-γ (Tyr783), Phospho-AKT (Ser473), Phospho-ERK (Thr202/Tyr204) (Cell Signaling, Danvers, MA, USA), and ACTIN (MP Biomedicals, Santa Ana, CA, USA). Horseradish peroxidase (HRP)-conjugated mouse (GeneTex, Irvine, CA, USA) and rabbit (Cell signaling) secondary antibodies were detected with ECL reagent (Thermo Fisher Scientific) and a ChemiDoc XRS+ System (Bio-Rad, Hercules, CA, USA).

Cells were lysed in RIPA lysis buffer (Pierce, Waltham, MA, USA) with fresh protease inhibitors (Pierce), and the lysate was quantified using the Micro BCA Protein Assay Kit (Pierce). Samples were boiled for 5 min in 4x LDS Sample Buffer (Invitrogen, Waltham, MA, USA) and separated by SDS-PAGE. Separated proteins were transferred to polyvinylidene difluoride membrane using the Mini Trans-Blot Electrophoretic Transfer Cell (BioRad, Hercules, CA, USA) for 90 min at 100 V. The following antibodies were used in Blocking Buffer (Thermo Scientific, Waltham, MA, USA): LC3A/LC3B (Invitrogen PA1-16931) at 1:500, SQSTM1 (Invitrogen PA1-27247) at 1:500, γH2AX (Novus NB100-384) at 1:1000, and Actin (Invitrogen MA1-744) at 1:1000. The membrane was washed with dPBST (Corning, +0.1% Tween) 3 times for 15 min each and incubated with secondary antibody (LC3A/B/γH2AX/p62: IRDye 800CW, Goat Anti-Rabbit; Actin: IRDye 680RD, Goat Anti-Mouse) at a 1:20,000 dilution in Blocking Buffer with 0.01% SDS and 0.1% Tween-20. Blots were visualized using a LiCOR imager and analyzed with Image Studio software.
Image studio was used to quantify relative expression of proteins in the Western blots by dividing the A.U. for key genes with the corresponding Actin control and were subsequently normalized to their respective experimental controls. Original blots can be found at Figures S9–S14.