Uvp chemstudio instrument

Protein lysates were prepared in RIPA lysis buffer, separated by 12% SDS-PAGE gel, and transferred to a nitrocellulose membrane (Cytiva, Marlborough, MA, United States) using a semi-dry method. Membranes were then probed overnight with the following primary antibodies against CDK6 (1:1,000; Santa Cruz; Cat #sc-7961), Cyclin A2 (CST, Danvers, MA, United States; Cat #4656T), PLK1 (CST; Cat #4513T), GAPDH (1:2,500; Invitrogen; Cat #437000), total Rb (1:500; Santa Cruz; Cat #sc-102), and phosphorylated Rb (1:1,000; CST; Cat #8516T for Ser807/811, #9307T for Ser780, and #9301T for Ser795). HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG (1:5,000; CST; Cat #7076S and #7074, respectively) were used as secondary antibodies. Blots were imaged using a UVP ChemStudio instrument (Analytik Jena, Beverly, MA, United States). Uncropped blots were shown in Suppl. Fig. 1.

Cell protein lysates were extracted using RIPA buffer (Sigma) including fresh protease and phosphatase inhibitors and standard western blotting protocol was performed as described before^{62 (link)}. For the EV marker analysis, 1 × 10¹¹ sEVs mL⁻¹ was loaded on the SDS gel. Primary antibodies: Anti-AnnexinA2 (Genscript A01471, 1/1000 dilution), anti-β-Actin (Abcam ab6276, 1/5000 dilution), anti-CD-9 (System Biosciences EXOAB-CD9A-1, 1/10000 dilution), anti-CD44 (Cell Signaling #3570, 1/1000 dilution), anti-CD63 (System Biosciences EXOAB-CD63A-1, 1/10,000 dilution), anti-CD81 (System Biosciences EXOAB-CD81A-1, 1/10,000 dilution), anti-Fibronectin (Abcam ab2413, 1/1000 dilution), anti-HSP-70 (System Bisociences EXOAB-HSP70A-1,1/10,000 dilution), anti-NEFL (Cell Signaling #2837, 1/1000 dilution), anti-OLIG2 (Genscript A01474, 1/1000 dilution), and anti-PTEN (Cell Signaling #9188, 1/1000 dilution). Secondary antibodies used: Polyclonal Goat Anti-Rabbit/Mouse Immunoglobulins/HRP (Dako P0447/8, 1/3000 dilution) antibodies and Anti-Rabbit Immunoglobulins/HRP (ExoAb antibody Kit, System Biosciences EXO-AB-HRP, 1/3000 dilution). Chemiluminescence was observed using a UVP Chemstudio instrument (Analytik Jena) and the Vision Works software. All experiments have been repeated at least three times.

Protein lysates were prepared in RIPA lysis buffer, separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane (Cytiva, Marlborough, MA, USA; Cat #10600012). Then, membranes were incubated overnight with the following primary antibodies against type I collagen (1:500, AbCam, #ab34710), α-SMA (1:500, Abcam, #ab5694), Erk1/2 (1:1000, CST, #9102S), p-Erk1/2 (1:1000, CST, #4370S), Smad2 (1:1000, CST, #5339S), p-Smad2, (1:1000, CST, #3108S) and GAPDH (1:2000, Invitrogen, #437000). HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG (1:5000, CST, #7076S and #7074, respectively) were used as secondary antibodies. Blots were scanned using a UVP ChemStudio instrument (Analytik Jena, Beverly, MA, USA). Uncropped blots are provided in Figure S5.