Sc 7313

Immunohistochemical staining was performed using freshly cut 5 micron sections from each site shipped to Stanford University and a commercial antibody for MUC1 (1:50 dilution; SC-7313, Santa Cruz Biotechnology) [20 (link)]. The digital image documentation of all stained slides was performed using the Leica SCN400 scanning system with the SL801 autoloader (Leica Microsystems; Concord, Ontario, Canada) at magnification equivalent to 40x. The images were transferred into the SlidePath digital imaging hub (DIH; Leica Microsystems). In parallel, separate TMA sections were stained with hematoxylin and eosin (H & E) and high molecular weight keratins (HMWK, 34bE12, Dako); these sections were scored for the presence of cancer in each core on the TMA as described previously [21 (link)–26 (link)]. A single pathologist (LF) scored MUC1 protein staining only in cores in which cancer was present as determined using the H & E and HMWK.
The immunohistochemical staining intensity for MUC1 was defined as absent, weak (faint cytoplasmic staining of scattered cells), moderate (intermediate or heterogeneous cytoplasmic staining in tumor cells), or strong (dense cytoplasmic staining of nearly all tumor cells) as shown in Fig 1.

Total protein was isolated from cells by using lysis buffer (P0013B; Beyotime, China). For high-throughput protein detection, we cut the membrane corresponding to the protein molecular weight prior to incubation with antibodies. Immunoblotting was performed with primary antibodies against MUC1 (sc-7313; Santa Cruz, USA; 1:200), ITGA2 (ab133557; Abcam, USA; 1:500), ITGA3 (ab131055; Abcam, USA; 1:500), ERK1/2 (4695; CST, USA; 1:1000), phosphorylated ERK1/2 (Thr202/Tyr204) (4370; CST, USA; 1:500) and GAPDH (60004-1-Ig; Proteintech, China; 1:5000) as the control and followed by the secondary antibodies (CW0102S and CW0103S; CWbiotech, China; 1:5000). The specific bands were visualized with super enhanced chemiluminescence (ECL) detection reagent (180–5001; Tanon, China).

Protein lysates from cells were extracted and measured as described in the Western blot section, and 600 µg of cell lysate protein was incubated for 3 h at 4 °C with 4 µg of biotinylated lectin VVA (EY Laboratories). Upon adding of 20 µl of streptavidin-agarose (Sigma Aldrich, Oakville, ON Canada), samples were incubated for an additional 2 h at 4 °C with rotation. Lectin/glycoprotein complexes were collected by brief centrifugation (1400 rpm, 5 min), and washed 3× with lysis buffer, followed by one wash with PBS. Glycoproteins were released from the complexes by boiling in 30–50 µl SDS–PAGE sample buffers (5 min). The glycoproteins were resolved by SDS–PAGE as described in the Western blot section, then immunoblotted to detect MUC1 (sc-7313, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) and FN1 (sc-69681, 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) protein expression.