Ab32373

The total protein was obtained using extraction kit (KGP250, KeyGen BioTECH, Nanjing, China), followed by the quantification using BCA protein concentration detection kit (KGA902, KeyGen BioTECH, Nanjing, China). By applying gel preparation kit (KGP113, KeyGen BioTECH, Nanjing, China), sodium dodecyl sulfate- (SDS-) PAGE was conducted followed by the membrane transfer. Thereafter, WB was performed, using anti-NIS (24324-1-AP, Proteintech Group, Inc., China), anti-Rap1GAP (ab32373, Abcam, UK), anti-TGF-β1 (ab215715, Abcam, UK), anti-Foxp3 (bs10211R, Bioss, China), anti-TβR1 (bs0638R, Bioss, China), anti-p-Smad3 (ab52903, Abcam, UK), anti-Smad3 (ab40854, Abcam, UK), anti-bcl-2 (ab182858, Abcam, UK), and anti-Bax (ab182733, Abcam, UK) with dilution rates of 1 : 1000, 1 : 10000, 1 : 1000, 1 : 1000, 1 : 1000, 1 : 2000, 1 : 1000, 1 : 2000, and 1 : 2000, respectively. After incubation with the secondary antibody, the membrane was colorized and imaged using G: BOX chemiXR5 (syngene, UK).

The protein levels of α-SMA, COL1A1, Smad2, p-Smad2, PED7A, PED4A, PKA, p-PKA, total Ras-proximate-1 (Rap1), Guanosine-5′-triphosphate (GTP)-Rap1, and exchange protein directly activated by cAMP 1 (Epac1), cAMP-response element-binding protein (CREB) and p-CREB were examined by immunoblotting following the methods described before (Liu et al., 2018 (link)) using the antibodies listed below: anti-α-SMA (ab5694, Abcam), anti-COL1A1 (ab34710, Abcam), anti-Smad2 (ab40855, Abcam), anti-p-Smad2 (ab53100, Abcam), anti-PDE4A (ab14607, Abcam), anti-PDE4B (ab170939, Abcam), anti-PDE4C (ab170939, Abcam), anti-PDE4D (ab171750, Abcam), anti-PKA (BS-0520R, Woburn, MA, United States), p-PKA (ab75991, Abcam), anti-GTP-Rap1 (ab32373, Abcam), anti-Rap1 (ab14404, Abcam), anti-Epac1 (ab109415, Abcam), anti-CREB (ab31387, Abcam), anti-p-CREB (ab32096, Abcam), and anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ab8245, Abcam) and then with HRP-conjugated secondary antibody. Enhanced chemilumescent (ECL) substrates (Millipore, MA, United States) were used for signals visualization using GAPDH as an endogenous protein for normalization.

Protein extraction from cells and tumor tissues and western blot analysis were performed as described as previously described [12 (link)]. Antibodies against ELK4 (abcam, ab86002), EZH2 (abcam, ab191250), H3K9me1 (abcam, ab16989), H3K9me2 (abcam, ab1220), H3K9me3 (abcam, ab8898), H3K27me2 (abcam, ab24684), H3K27me3 (abcam, ab6002), H3 (abcam, ab1791), E-cadherin (CST, 14472), EAF2 (CST, 14159), ADRB2 (abcam, ab182136), rap1GAP (abcam, ab32373), RUNX3 (abcam, ab224641), GAPDH (CST, 5174) and Notch1 (CST, 3608) were used.