Alexafluor 680 goat anti rabbit

Immunoblots were performed as described (Todd et al., 2011 (link)). Membranes were probed with the indicated primary antibodies and beta-actin (D6A8) as loading control. Goat anti-rabbit-AlexaFluor680 (Invitrogen) and goat anti-mouse-IRDye800 (Rockland Immunochemicals, Gilbertsville, PA) were used to detect membrane-bound antibodies. Signal was visualized using the LiCOR Odyssey imaging system and software (LiCOR, Lincoln, NE).

Western blotting was performed essentially as described previously^{6 (link)}. Briefly, Immobilon-FL PVDF membranes (Merck Millipore) were pre-activated with MeOH and used for wet blotting for 1 h at 100 V. Blocking was accomplished with casein blocking buffer (Sigma) for 30 min at room temperature while shaking on a roller shaker. Then, the primary antibody solution in PBS with 0.1% (v/v) Tween-20 and 1 × casein blocking buffer (antibody incubation buffer) was applied on the blot and incubated for 1 h at room temperature while shaking on the roller shaker. The primary antibodies used were streptavidin-IRDye 680LT (LI-COR Biosciences), polyclonal rabbit anti-HA (H6908, Sigma-Aldrich), mouse anti-actin (ab8224, Abcam) and a home-made rabbit anti-DARPin serum. Secondary antibodies were goat anti-rabbit Alexa Fluor 680 (Invitrogen), donkey anti-mouse IRDye800 CW (LI-COR Biosciences) and goat anti-rabbit IRDye800 CW (LI-COR Biosciences). When incubating with the fluorescently labelled streptavidin (IRDye 680LT), also 0.1% (w/v) SDS was included to reduce non-specific binding of streptavidin. Blots were either incubated with streptavidin-IRDye 680LT and anti-DARPin serum together, or with the anti-HA and anti-actin antibodies together. Detection was accomplished using an Odyssey 9120 imaging system (LI-COR Biosciences).

Ab109115 (Abcam) was used at a dilution of 1:100, and Mab2166 (Merck Millipore) was used at 1:1000 for immunofluorescence. S421 was a kind gift from Dr. Sandrine Humbert, (Institut Curie, Paris, France), and was used at 1:1000 for immunofluorescence. Anti-EGFR (New England Biolabs) was used at 1:50 for immunofluorescence and 1:1000 for western blots. Calreticulin (Abcam) was used at 1:500 for immunofluorescence, and α-tubulin was used at 1:5000 for western blots. Anti-CTIP2 (Abcam) was used at 1:500, and Anti-DARPP32 (Santa Cruz Biotechnology Inc.) was used at 1:200; both for immunofluorescence. Secondary antibodies; Goat-anti-Rabbit AlexaFluor 568 and Donkey-anti-Mouse AlexaFluor 488 (Life Technologies) were both used at 1:100 for immunofluorescence. Goat-anti-Rabbit AlexaFluor 680 (Life Technologies) and Goat-anti-mouse DyLight 800 were used for Western blots at 1:10,000 and 1:20,000, respectively.

Breast cancer cell lines BT-20, BT-549, HCC-1806, HCC-1937, HCC-38, MDA-MB-231, MDA-MB-436, and MDA-MB-468 were obtained from American Type Culture Collection (ATCC). Breast cancer cell line MFM-223 was purchased from Sigma. Sum-159 breast cancer cell line generation was previously described [33 (link)]. All these cells were cultured in Dulbecco’s modified Eagle’s medium/F-12 supplemented with 10% fetal bovine serum at 37° C and in a humidified incubator containing 5% CO₂.
Paclitaxel and carboplatin were obtained from Selleck Chemicals. Eribulin was acquired from D Anderson Cancer Center’s pharmacy (Houston, TX, USA). For in vitro studies AZD8186 and AZD5363 were obtained from Selleck Chemicals. For in vivo studies AZD8186 was obtained from AstraZeneca as a generous gift. Anti-mouse PD1 monoclonal antibody (clone: RPM1-14) was purchased from BioXcell. For in vitro studies, all drugs were dissolved in DMSO.
Immunoblotting antibodies purchased from Cell Signaling Technology (CST) include anti-phospho-AKT (S473) (#4060), anti-AKT (#9272), anti-phospho-S6K (T389) (#9234), anti-S6K (#9202), anti-phospho-PRAS40(T246) (#2997), and PRAS40 (#2610). Anti-β-actin antibody (#A5441) was purchased from Sigma. Secondary antibodies Goat-anti-Rabbit-Alexa Fluor-680 (#A21076) and Goat-anti-Mouse-Dyligh-800 (#610145-121) were purchased from Life Tech and Rockland Immunochemicals, respectively.