Complete mini cocktail

Nuclear and cytoplasmic protein was extracted from cells by suspension in a solution of Buffer A containing 10 mM HEPES pH 7.4, 1.5 mM MgCl₂, 10 mM NaCl, 0.1% NP-40 and a cocktail of protease inhibitors (Complete Mini Cocktail, Roche Diagnostics Ltd, Lewes, UK). The cells were lysed on ice for 10 min after which they were passed through a 21 gauge needle to ensure complete plasma membrane lysis. Nuclei were pelleted by centrifugation (12,000 x g at 4°C for 2 min) and the supernatant containing cytoplasmic protein was retained. Nuclei were washed in a solution of Buffer A (as specified above). The nuclei were lysed for 10 min on ice in Buffer A and sonicated for 30 sec to completely disrupt the nuclear membrane. Remaining cell debris was pelleted by centrifugation and the supernatant containing nuclear protein retained. Total protein content was determined by the Bradford protein method using the BCA protein assay kit (Pierce, Cramlington, UK). Protein (30 μg) was loaded onto a Tris-Glycine polyacrylamide gel (10%) and subsequently transferred to a nitrocellulose membrane. The antibodies used for Western blotting were β-tubulin (Abcam 1:1000), HOXD10 (Biorbyt 1:200), POU2F1 (Abcam 1:1000) and TATA-BP (Abcam 1:1000).

Western blot analysis was performed as described previously (Wang et al., 2010 (link)). Briefly, the collected samples of NPs, cell pellets or brain tissues were lysed in cold T-PER buffer containing 1% phosphatase inhibitors and complete mini cocktail (Roche, Switzerland). Protein concentrations were measured by the Bicinchoninic (BCA) assay kit (Pierce, China). Twenty micrograms of each protein sample were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, USA). Transblotted PVDF membranes were blocked with 5% BSA for 1 h and then incubated overnight with the indicated primary antibody in 1% BSA at 4 °C and followed by the secondary antibody conjugated with horseradish peroxidase. Blots were visualized using ECL-Plus according to the manufacturer’s instructions.

To prepare pancreas extracts, mice were euthanized, and pancreata (normal or pathological) were removed and frozen in liquid N₂. Pancreata were lysed in buffer L1 (50 mM Tris-HCl pH 8.0, 2% SDS, 10% glycerol) plus phosphatase (1 mM β-glycerol phosphate, 10 mM NaF, 1 mM sodium orthovanadate) and protease inhibitors (complete mini cocktail, ROCHE), using Lysing Matrix D (MP #6913–050). Lysate was heated at 95°C for 5 min, passed sequentially through 18G, 21G, 23G, and 25G syringes, sonicated for 10 min, and centrifuged at 13, 600 × g for 10 min. Protein (5 μg) was fractionated by 10% or 15% SDS-PAGE and analyzed by Western Blot using the following antibodies: anti-amylase (Sigma-Aldrich), anti-chymotrypsinogen (Biogenesis), anti-β-actin (Abcam), or anti–pyruvate kinase (Merck).

The first-void mid-stream urine sample of healthy volunteers was collected into plastic containers, and three protease inhibitors (cOmplete, Mini, Cocktail, Roche Applied Science, Mannheim, Germany) were added to 70 mL of urine. To remove contaminants, the urine samples were first centrifuged at 300× g (10 min) prior to a higher speed centrifugation step at 2000× g (10 min).
The urine samples were then clarified by vacuum filtration (TPP rapid Filtermax with 0.22 μm PVDF) before proceeding to a concentration step using a filter device (Centricon MWL 10,000 UFC701008, Merck Millipore, Darmstadt, Germany). For this concentration step, the precleared urine was centrifuged at 3220× g (Eppendorf centrifuge 5810R, Rotor A-4-62, Vaudaux-Eppendorf, Schönenbuch, Switzerland) according to the manufacturer’s instructions. The concentrate was eluted at a speed of 1000× g for 2 min.
The collected small EVs were stored at −20 °C for further analysis.
The urinary small EVs were further purified using qEV exosome isolation size-exclusion chromatography (SEC) columns (Izon Science, Oxford, UK) according to the manufacturer’s instructions, and the eluted fractions were analysed for exosome size and concentration using nanoparticle tracking analysis (NTA) as described below.

In total, 0.2–1 × 10⁶ CTLs were lysed using cold cell lysis buffer containing 50 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, and 0.25% sodium deoxycholate. Suppression of talin 1 was confirmed using an anti-talin 1 antibody (clone 8D4, Abcam). Talin head domain GFP fusion was detected using a polyclonal antibody against GFP (Invitrogen). Actin (clone AC-15, Sigma) or GAPDH (clone D16H11, Cell Signaling Technology) served as loading controls. For signaling assays, serum and IL-2 starved OT-1 CTLs were incubated with streptavidin polystyrene beads (Spherotech) coated with H-2K^b-OVA and ICAM-1 at a 1:1 ratio for various times at 37 °C and immediately lysed in 2× cold lysis buffer containing phosphatase inhibitors (1 mM NaF and 0.1 mM Na₃VO₄) and protease inhibitors (cOmplete mini cocktail, EDTA-free, Roche). Activation of PI3K and MAP kinase signaling was assessed by immunoblot for pAkt (Phospho-Akt (Ser473) Ab; Cell Signaling Technology) and pErk1/2 (Phospho-Thr202/ Tyr204; clone D13.14.4E; Cell Signaling Technology). Additional information about antibodies may be found in Supplementary Table 1. Uncropped images of blots are provided in Supplementary Fig. 12.

Madin-Darby canine kidney cells were seeded in 12-well plates and were infected with influenza A virus. At 9 hpi, cells were suspended in 100 μl of TNE buffer (50 mM Tris pH 7.5, 1 mM EDTA, and 150 mM NaCl) containing 1 mM DTT and protease inhibitors (Complete Mini cocktail, Roche). After brief sonication, the cells were treated with 1% TX-100 at 4°C or 37°C for 30 min and were centrifuged at 17,400 × g for 30 min at 4°C to separate the soluble and insoluble fractions. The fractions were analyzed by western blotting with anti-GFP mAb (clone GSN149, Sigma-Aldrich) and anti-caveolin1 rabbit Ab (H-20, Santa Cruz).
For coimmunoprecipitation, the fractions were mixed with 10 μg of anti-HA mAb12-1G6 for 90 min at 4°C and were subsequently mixed with protein G-Sepharose beads (GE Healthcare) pretreated with 1% BSA at 4°C for 60 min. Following several washes with TNE buffer containing 0.1% TX-100, the beads were boiled in SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE followed by western blotting with anti-HA mAb12-1G6 and anti-GFP mAb. Chemiluminescent signals were detected using an Image Quant LAS500 (GE Healthcare) and were quantified using ImageJ software.