Ab72704

Co-IP was carried out as reported previously^{56 (link)}, using 10 µg of antibodies for ARMC12 (ab72704, Abcam), RBBP4 (ab1765, Abcam), EZH2 (ab191250, Abcam), SUZ12 (ab12073, Abcam), FLAG (ab125243, Abcam), and HA (ab9110, Abcam). After releasing from bead-bound complex, protein levels were measured by western blotting. Uncropped images of blots were shown in Supplementary Fig. 10. For proteomic analysis, proteins were digested by trypsin, and peptides were extracted according to Invitrogen protocol. LC-MS/MS detection was undertaken on a hybrid quadrupole-TOF LC/MS/MS mass spectrometer (TripleTOF 5600+, SCIEX, Redwood City, CA). For each scan cycle, one full-scan mass spectrum (with m/z ranging from 350 to 1500 and charge states from 2 to 5) and subsequent 40 MS/MS events were carried out. Analysis of MS/MS-acquired data was performed using ProteinPilot Software 5.0 and amino acid sequences in UniProt human protein database.

Protein of tumor cells or tissues was prepared using 1× cell lysis buffer (Promega). Western blotting was performed as described previously^{52 (link)–55 (link)}, with antibodies specific for ARMC12 (ab72704, Abcam, Cambridge, MA, 1:1000 dilution), RBBP4 (ab1765, Abcam, 1:1000 dilution), EZH2 (ab191250, Abcam, 1:1000 dilution), SUZ12 (ab12073, Abcam, 1:500 dilution), H3K27me3 (ab6002, Abcam, 1:1000 dilution), HA (ab9110, Abcam, 1:1000 dilution), FLAG (ab125243, Abcam, 1:1000 dilution), CADM1 (ab3910, Abcam, 1:500 dilution), EGLN3 (ab30782, Abcam, 1:1000 dilution), HRK (ab45419, Abcam, 1:1000 dilution), HS6ST3 (SAB2101082, Sigma, 1:500 dilution), SMAD9 (ab115900, Abcam, 1:500 dilution), histone H3 (ab1791, Abcam, 1:1000 dilution), and GAPDH (ab8245, Abcam, 1:1000 dilution). Uncropped images of blots were provided in Supplementary Fig. 10.

Immunohistochemical staining was undertaken as described previously^{52 (link),53 (link),57 (link),62 (link)}, with antibodies specific for ARMC12 (ab72704, Abcam, 1:200 dilution) or RBBP4 (ab1765, Abcam, 1:200 dilution). To assess the reactivity degree, ten different high power fields (×400) for each specimen were blindly evaluated. The staining intensity was evaluated on a range from 0 to 3 (0, negative; 1, weakly positive; 2, moderately positive; 3, strongly positive), while percentage of positive cells were evaluated ranging from 0 to 4 (0, negative; 1, positive in 1−25%; 2, positive in 26−50%; 3, positive in 51−75%; 4, positive in 76−100%). Based on the products of staining intensity multiplied by percentage of positive cells, the results of immunohistochemistry were classified into negative (−, 0–1), mildly (+, 2–3), moderately (++, 4–8), and strongly positive (+++, 9–12). The moderate (++) or strong (+++) reactivity was defined as high expression, while negative (−) or mild positive (+) reactivity was defined as low expression.