Anti hdlbp

Immunoblotting was performed as previously described.¹⁹ (link)^,34 (link) The β-actin antibody was used to normalize protein expression. For CHX chase assays, cells were treated with 10 μmol CHX for 24 hours after transfection and collected at the indicated time points, and cell lysates were subjected to immunoblotting. Anti-HDLBP (Proteintech, 15406-1-AP, Wuhan, China, 1:800), Anti-RAF1 (Cell Signaling Technology, #53745, Danvers, MA, 1:800), Anti-β-actin (Proteintech, 66009-1-Ig, 1:2500), Anti-Lamin B1 (Proteintech, 12987-1-AP, 1:2500), Anti-Flag (Proteintech, 20543-1-AP, 1:2000), Anti-HA (Proteintech, 51064-2-AP, 1:3000), Anti-His (Proteintech, 66005-1-Ig, 1:2500), Anti-Myc (Proteintech, 16286-1-AP, 1:800), Anti-TRIM71 (Proteintech, 55003-1-AP, 1:800), Anti-NEDD4L (Proteintech, 13690-1-AP, 1:800), Anti-p-MEK (Cell Signaling Technology, #8727, 1:500), Anti-MEK (Cell Signaling Technology, #9154, 1:500), Anti-p-ERK (Cell Signaling Technology, #4695, 1:500), Anti-ERK (Cell Signaling Technology, #4377, 1:500), Anti-MEKK1 (Proteintech, 19970-1-AP, 1:500), Anti-RAF1^S338 (Cell Signaling Technology, #9427, 1:500), Anti-RAF1^S259 (Cell Signaling Technology, #9421, 1:500), Anti-RAF1^S621 (Abcam, ab157201, Cambridge, UK, 1:500), Anti-RAF1^Y341 (Abcam, ab59223, 1:500), and Anti-RAF1^S289/296 (Cell Signaling Technology, #9431, 1:500) were used.

The CoIP assay was performed using CoIP kits (Abs955, Absin, Shanghai, China) according to the manufacturer’s recommended protocol. Briefly, the indicated cells were homogenized in immunoprecipitation lysis buffer (20 mM Tris-HCl pH 7.5, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM DTT, 1 mM cocktail, 1 mM phosphoSTOP, 1 mM NEM, and 1 mM NAM). Five hundred micrograms of extracts were incubated with the indicated primary antibody or IgG as a negative control for 4 hours and Protein A/G-Sepharose beads for 2 hours at 4 °C. After extensive washes with phosphate buffered saline, the immunoprecipitates were used in the subsequent assays. Anti-HDLBP (Proteintech, 15406-1-AP, Wuhan, China, 1:100), Anti-RAF1 (Cell Signaling Technology, #53745, MA, USA, 1:50) and Anti-IgG (Cell Signaling Technology, #3900, MA, USA, 1:500) were used.

Immunofluorescence staining was performed as previously described.¹⁹ (link) Anti-HDLBP (Proteintech, 15406-1-AP, 1:100) and Anti-RAF1 (Santa Cruz Biotechnology, sc-7267, Dallas, Tx, 1:50) were used.