Ab14404

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.

The interaction of LRPAP1, LRP1 and IFNAR1 was examined by immunoprecipitation using a protocol described as followed. HEK293T cells were cultured at around 90% confluence in a 100 mm tissue culture dish. Cells were washed with 10 ml of PBS and harvested in 0.5 ml of ice-cold lysis buffer (0.15 mM NaCl, 0.05 mM tris-HCl, pH 7.4, 1% SDS, 1% NP-40) and Protease Cocktail (Roche) and PhoSTOP (Roche). The total extract was pre-cleared with 10 μg normal rabbit IgG (Cell signaling, 2729 S) and 20 μl of protein A/G plus-agarose (Santa Cruz Biotechnology) at 4 °C on a rotor for 1 h. After gently removed protein A/G agarose beads by centrifugation at 500 rcf for 2 min, 0.2 mg of total extract was immunoprecipitated with the indicated antibodies overnight at 4 °C on a rotor. Approximately 20 μl of protein A/G plus-agarose (Santa Cruz Biotechnology) was added at 4 °C for one hour. Then, the beads were washed three times with washing buffer (lysis buffer contained 0.1% NP-40). The pellet was suspended with 40 μl PBS and 10 μl of 5× SDS-PAGE loading buffer and the samples were boiled for 10 min. The supernatant was collected and 20 μg of lysate was loaded onto 10% acrylamide gene electrophoresis and immunoblotting was performed using antibodies against IFNAR1 (Abcam, ab45172), LRP1 (Abcam, ab92544), LRPAP1 (Abcam, ab14404) as described before.