Ab40776

Immunoblot analyses of lung tissue were performed on total lysates as described by Ding et al. (2019) (link) using phospho-signal transducer and activator of transcription (STAT) 3 (p-STAT3; catalog number, ab76315; Abcam, Cambridge, United Kingdom), STAT3 (ab68153; Abcam), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA; ab40776; Abcam), phospho-mitogen-activated protein kinase (MAPK) 1 (p-MAPK1; ab201015; Abcam), MAPK1 (ab184699; Abcam), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 60004-1-Ig, Proteintech) as primary antibodies and then incubation with the corresponding secondary antibodies. Protein bands were detected by an electrochemiluminescence reagent (NCM Biotech, Beijing, China). The intensity of protein bands was analyzed using Image-Pro Plus (Media Cybernetics, Rockville, ML, United States) and presented as the ratio to GAPDH.

Plasmids with His‐tagged ubiquitin, Flag‐NEDD4L, and HA‐PIK3CA were transfected into HEK293T cells. In order to enrich the ubiquitinated protein, the cell lysate was incubated with Talon beads (GE Healthcare). After incubating for 2 h at 4°C, ubiquitinated proteins were eluted with 20 ml of eluent (5 × SDS buffer, 0.05 M ethylenediaminetetraacetate [EDTA], and 1% SDS lysis buffer). Ubiquitinated proteins were subjected to immunoblotting with PIK3CA (ab40776; 1: 1000; Abcam) or HA antibody (ab9110; 1: 2000, Abcam).

The rats were perfused with 200 mL of saline followed by 200 mL of 0.1 M phosphate buffer (pH 7.3) containing 4% paraformaldehyde. The L₄-L₅ spinal cord was removed, postfixed in 4% paraformaldehyde for 24 h, and allowed to equilibrate in 30% sucrose in phosphate-buffered saline (PBS) overnight at 4°C. Transverse spinal sections (4–6 μm) were cut using a cryostat and collected in 0.01 M PBS, pH 7.3. After washing with PBS, the tissue was penetrated with 0.3% Triton X-100 and primary antibodies for rabbit anti-rat Iba1, BDNF, p-ERK, and PI3K (rabbit anti-Iba1, ab178680, 1 : 100, Abcam, USA; rabbit anti-BDNF, ab108319, 1 : 500, Abcam, USA; rabbit anti-p-ERK, #4370, 1 : 200, Cell Signaling Technology, USA; rabbit anti-PI3K, ab40776, 1 : 200, Abcam, USA). Next, the slides were covered with secondary antibodies containing 1 μM 4′-6-diamidino-2-phenylinedole (Sigma, USA). Some sections stained for BDNF, p-ERK, and PI3K were double labeled using cell-type-specific Abs for microglia (Iba1). The nuclei were stained with DAPI (5 μg/mL; Beyotime, USA). Fluorescence signal was detected using a fluorescence microscope (Olympus, Japan), images were captured, and signal co-localization was measured using MetaMorph (Molecular Devices, USA). The area fraction was quantified using Image J software (Rawak Software, Inc., Germany).

Differentiated C2C12 myotubes grown in 25-cm² culture-flasks (containing 5 ml DMEM) were treated with KPC-exosomes (0 μg, 1 μg, 2 μg, 4 μg), MPDC-exosomes (4 μg) and insulin (10⁻⁷ M: 0 min, 5 min, 15 min, 30 min).
Procedures for protein extractions and immunoblots were carried out according to standard protocols. Nuclear and cytoplasmic proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (78833, Thermo Scientific, Waltham, MA, USA). Primary antibodies against PTEN (ab32199, Abcam), IRR (ab40782, Abcam), IRS-1 (#3407, CST), PI3 Kinase p110α (ab40776, Abcam), PI3 Kinase p110β (ab151549, Abcam), PI3 Kinase p110γ (#5405, CST), PI3 Kinase p110δ (ab109006, Abcam), Phospho-Akt (Ser473) (#4060, CST), Akt (#2920, CST), FOXO1 (#2880,CST), Phospho-FoxO1(Ser256)(#9461, CST), Glut4 (#2213, CST), Lamin A + C (ab108922, Abcam), β-actin (sc-47778, Santa Cruz Biotechnology), and LC3B (ab192890, Abcam) were used.