Mab3540

Cells were collected and lysed using NP40 lysis buffer for 30 minutes on ice with constant agitation. Lysates were centrifuged at 12,000 x g for 5 minutes at 4°C, supernatants were collected, and then protein content was quantified (#500-0006, Biorad, Hercules, CA, USA). Protein was separated on 10% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk, then probed with primary antibodies against Lamin A (MAB3540, Millipore, Billerica, MA, USA), Progerin (#05-1231, Millipore), GFP (AB3080, Millipore), Oct4 (MAB4401, Millipore), and Notch2 (Ab8926, Abcam, Cambridge, MA, USA) in 5% milk for 1h at room temperature. Membranes were then washed in TBS-tween and incubated in 5% milk supplemented with HRP-linked secondary antibodies (SC2005, goat anti-mouse IgG-HRP; SC2060, goat anti-mouse IgG1-HRP; SC2004, goat anti-rabbit IgG-HRP; SC2020, donkey anti-goat IgG-HRP, Santa Cruz Biotechnology, Dallas, Texas, USA). Protein bands were detected by enhanced chemiluminescence using the ECL Plus kit (RPN2132, GE Life Sciences, Pittsburg, PA, USA). Total protein stained with coomassie brilliant blue (Biorad, 161-0400) was used as a loading control.

MIAMI cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, rinsed 3× 5 minutes in PBS, and stored in sterile water at 4°C. Cells were permeabilized with 0.1% triton-x for 5-10 minutes at room temperature, blocked in 3% PBS-BSA for 30 minutes at room temperature and then stained with primary antibodies against GFP (AB3080, Millipore), Notch 2 (Ab8926, Abcam), Oct4 (MAB4401, Millipore), Lamin A (MAB3540, Millipore), Progerin (#05-1231, Millipore), Ki-67 (KI67-MM1-L-CE, Leica Biosystems, Buffalo Grove, IL,USA) for 1h at room temperature. Rinsed cells were incubated with fluorescently labeled secondary antibodies for 1h at room temperature. Coverslips were mounted on glass slides using DAPI mounting media. Cells not plated on coverslips were incubated with DAPI antibody for 15 minutes at room temperature prior to imaging.

Expression of proteins were detected using antibodies against mouse lamin A/C (SAB4200236, Sigma‐Aldrich, St. Louis, MO, USA), human lamin A (MAB3540, Millipore, Burlington, MA, USA), Sarcolipin (ABT13, Millipore), β‐tubulin (ab179513, Abcam, Cambridge, UK), FLAG (F7425, Sigma‐Aldrich), HA (H6908, Sigma‐Aldrich), lamin B1 (ab133741, Abcam), lamin B (sc‐6217, Santa Cruz, Dallas, TX, USA), Calnexin (MAB3126, Millipore), Calreticulin (PA3‐900, Thermo Fisher Scientific), SERCA2 (4388, Cell Signaling, Danvers, MA, USA), β‐ACTIN (A5441, Sigma‐Aldrich), Grp78 (ab108613, Abcam), Chop (2891, Cell Signaling), eIF2α (2103, Cell Signaling), phospho‐eIF2α (3398, Cell Signaling), Atf4 (SC‐200, Santa Cruz), IRE1α (3294, Cell Signaling), phospho‐IRE1α (PA1‐16927, Thermo Fisher Scientific), and Gapdh (GTX100118, GeneTex, Hsinchu, Taiwan).