Anti gapdh

Primary antibodies used included anti-Cav2.1 (1:500, Alomone Labs), anti-GFP rabbit polyclonal (1:2000, Invitrogen/Thermo Fisher Scientific), anti-GAPDH (1:3000, Novus Biologicals), anti-transferrin receptor (1:1000, Invitrogen/Thermo Scientific), anti-CACNA2D1 (1:1000, Abcam), anti-Cavβ₄ (1:1000, Neuromab), anti-AnkB (1:500, Thermo Fisher Scientific), and anti-syntaxin 1A (1:5000, Millipore Sigma). Secondary antibodies used included horseradish peroxidase (HRP)-conjugated AffiniPure donkey anti-rabbit immunoglobulin and donkey anti-mouse IgG (all at 1:4000; Jackson ImmunoResearch).

Cell protein was extracted using RIPA lysis buffer containing protease and phosphatase inhibitor cocktails (cat. no. 539134, Millipore and cat. no. 78420, Thermo Fisher Scientific), and the protein concentration was determined using the protein assay kit (cat. no. 23200, Thermo Fisher Scientific, United States). Separation of proteins was subjected to the WES system (ProteinSimple, United States) and 12–230 kDa separation modules were used. For target proteins detection, the following antibodies were used: anti-NF- kB p65 (Cell Signaling Technology, #6956), anti-phospho- NF- kB p65 (Ser536) (Cell Signaling Technology, #3033), anti-Nrf2 (Cell Signaling Technology, #12721), anti-IL-6 (Abcam, ab9324), and anti-GAPDH (Novus Biologicals, NB300-325).

ELISA kits were obtained from BioLegend: IL‐4 (Cat. No. 431104) and IL‐5 (Cat. No. 431240). Antibodies used in our experiments were as follows: Anti‐cleaved caspase 3 (Cat. No 9664, clone 5A1E) and anti‐sirtuin 1 (SIRT1) (Cat. No. 3931 clone D60E1) were obtained from Cell Signaling Technology; anti‐MFG‐E8 (Cat. No. 377356, clone H‐3) was obtained from Santa Cruz; anti‐PPAR‐γ (Cat. No. ET1702‐57 clone JF101‐4) was obtained from HuaAn Biotechnology; anti‐PGRN (Cat. No. NBP1‐32076, polyclonal) was obtained from Novus Biologicals; anti‐GAPDH (Cat. No. 10494‐1‐AP polyclonal) was obtained from Proteintech and anti‐PE‐F4/80 Cat. No. 123109, clone BM8) was obtained from BioLegend. The following reagents were purchased from Sigma‐Aldrich; ovalbumin (OVA) (Cat. No. A5503‐50G); GW9662 (Cat. No. M6191); dexamethasone (Cat. No. D4902) and hydrogen peroxide (H₂O₂) (Cat. No. 323381). The following reagents were purchased from Thermo Fisher: CellTrace CFSE (Cat. No.C34570); lipofectamine 2000 (Cat. No. 11668019); Inject Alum (Cat. No. 77161). Recombinant M‐CSF (Cat. No. 315‐02) was from PeproTech. Iso plus (Cat. No. 9109) and PrimeScript RT reagent kit (Cat. No. RR037A) were purchased from Takara. siRNA was purchased from GenePharma. All primers and SYBR Green were purchased from Tsingke Biotechnology. The TUNEL kit (Cat. No. G1501) was obtained from Servicebio.

Western blotting was performed using anti-4-hydroxynonenal (4-HNE; Abcam), anti-peroxiredoxin (Prx-SO₃, Abcam), anti-MnSOD (Calbiochem, San Diego, CA), anti-copper-zinc superoxide dismutase (CuZnSOD; Chemicon, Temecula, CA), anti-histone H1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2)^{40 (link)}, anti-Opa1 (BD Bioscience, Franklin Lakes, NJ, USA), anti-Drp1 (Cell Signaling Technology, Danvers, MA, USA), anti-Bax (5B7; Santa Cruz), anti-Bcl-2 (Cell Signaling Technology), anti-cytochrome c (BD Bioscience), anti-cleaved caspase-3 (Merck Millipore, Darmstadt, Germany), anti-β-actin (Sigma), and anti-GAPDH (Novus, Littleton, CO, USA) antibodies.

The protein expression of odontogenic-related genes, including dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP-1), alkaline phosphatase (ALP), and runt-related transcription factor 2 (Runx2), was detected by Western blot analysis. The DPSCs were appropriately cultured in an odontogenic medium containing either 20 µg/mL CM-EV or OM-EV for 7 and 14 days as explained above. Cells were then collected and lysed in RIPA buffer (KeyGen BioTECH, Nanjing, China) containing a 1% protease inhibitor cocktail (CWBIO, China). The protein concentration was determined by employing the BCA assay kit (CWBIO). The proteins of each group were separated by 10% SDS-PAGE (GenSpirt, China) and transferred to PVDF membranes (Millipore, USA). After blocking with 5% fat-free milk, the PVDF membranes were subsequently incubated overnight at 4 °C with primary antibodies anti-DSPP (1:500, Novus, USA), anti-DMP-1 (1: 500, Bioss), anti-Runx2 (1:1000, Novus), anti-ALP (1:1000, Novus), anti-GAPDH (1:1500, Novus), followed by incubation with secondary antibody (1:5000, GNI, Japan). An advanced chemiluminescence detection system (Millipore) was utilized to detect immunoblots with an imaging system (ChemiDoc, Bio-Rad, China). The bands’ intensities were suitably measured via ImageJ and normalized to GAPDH.

Immunoblotting was performed essentially as previously described (13 (link)). In brief, equal amounts of proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). Membranes were blocked for 1 h with PBS containing 5% low fat milk powder (Roth, Karlsruhe, Germany) followed by overnight incubation at 4°C with primary antibodies anti-IL-1β 1:20,000 (kindly supplied by the National Cancer Institute, Frederick, MD), anti-Anx2 1:1,000 (Abnova, Taipai, Taiwan), anti-Arrb1 1:500 (Zymed, Carlsbad, CA), anti-Arrb2 1:2,000 (Abnova), anti-β-actin 1:100,000 (Sigma-Aldrich) or anti-GAPDH 1:10,000 (Novus Biologicals, Littleton, CO) in blocking solution. After extensive washing, the membranes were further incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako, Glostrup, Denmark). For the detection of bound peroxidase, SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) was used. Only β-actin and GAPDH were revealed with the less sensitive Lumi-Light substrate (Roche, Mannheim, Germany). To quantify the signal, densitometric analyses were performed using a digital gel documentation system (Biozym, Hessisch Oldendorf, Germany).