Ab133741

Western blotting was performed as previously described^{26 (link)}. Primary antibodies were polyclonal antibodies to xCT (NB300-318, Novus Biologicals, Littleton, CO, USA), GPX4 (NBP2-75511, Novus Biologicals), ferritin heavy chain 1 (FTH1, #3998, Cell Signaling Technology, Danvers, MA, USA), p53 (#9282 S, Cell Signaling Technology), NRF2 (NBP1-32822, Novus Biologicals), β-actin (A5441, Sigma-Aldrich), or Lamin B1 (ab133741, Abcam). Membranes were washed three times for 10 min in TBS with 0.1% Tween-20 then incubated in buffer A containing a 1:1000 dilution of horseradish peroxidase-conjugated goat-anti rabbit, or anti-mouse IgG (GenDEPOT, Barker, TX). ImageJ software (NIH, Bethesda, MD, USA) was used to measure band intensities, and changes in treated groups relative to control cells or tissues were used for analysis.

The total cellular extracts of LECs were prepared in ice-cold radioimmune precipitation buffer (RIPA buffer), and Western analysis was carried out as described previously [7 (link),70 (link),71 (link),72 (link)]. The membranes were probed with anti-Nrf2 (sc-722, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Nrf2 (ab62352, Abcam^®, Cambridge, MA, USA), Anti-Klf9 (ab177158, Abcam^®, Cambridge, MA, USA), Anti-Klf9 (sc-12996, Santa Cruz Biotechnology, Dallas, TX, USA), Anti-Prdx6 antibody (LF-PA0011, Ab Frontier, South Korea), Tubulin (ab44928, Abcam^®, Cambridge, MA, USA) or β-actin (A2066, Sigma-Aldrich, St. Louis, MO, USA) or Lamin B1 (ab133741, Abcam^®, Cambridge, MA, USA) as an internal control to monitor levels of protein expressions. After secondary antibody (sc-2354 and sc-2768, Santa Cruz Biotechnology, Dallas, TX, USA) incubation, protein bands were visualized by incubating the membrane with luminol reagent (sc-2048; Santa Cruz Biotechnology, Dallas, TX, USA). Finally, images (bands) were recorded with a FUJIFILM-LAS-4000 luminescent image analyzer (FUJIFILM Medical Systems Inc., Hanover Park, IL, USA).

Protein samples derived from mouse skins and cells were extracted by RIPA lysis buffer containing phosphatases and proteases inhibitor cocktails (Roche, USA). Protein concentration was determined by BCA protein assay kit (Pierce, USA). For immunoblotting analysis, protein samples were subjected with SDS-polyacrylamide gel electrophoresis and proteins were transferred to PVDF membranes (Millipore). After being blocked, and incubated with primary antibodies and indicated HRP-coupled secondary antibody, membranes were visualized with ECL and images were captured using the Bio-Rad system. Band intensities were detected, normalized, and quantified with the Image Lab 5.0 software (Bio-Rad). The following antibodies were used in this research: NAT10 (Abcam, ab194297, 1:1000 dilution), STAT3 (CST, 30835, 1:1000 dilution), p-STAT3 (Abcam, ab76315, 1:1000 dilution), p-p65 (CST, 3033, 1:1000 dilution), p65 (CST, 8242, 1:1000 dilution), p-FAK(CST, 8556, 1:1000 dilution), FAK (Abcam, ab40794, 1:1000 dilution), Lamin B1 (Abcam, ab133741, 1:1000 dilution), poly-Ubiquitin (CST, 3936, 1:1000 dilution), Fibronectin(CST, 26836, 1:1000 dilution), α-SMA(Proteintech, 14395-1-AP, 1:1000 dilution), α-Tubulin (Beyotime, AT819, 1:5000 dilution), and GAPDH (Beyotime, AG019, 1:5000 dilution).

Primary antibodies against MARCO (ab239369), TRIF (ab13810), NF‐κB (ab16502), TNF‐α (ab183218), Lamin B (ab133741) and GAPDH (ab181602) as well as the secondary antibody of goat anti‐rabbit were purchased from Abcam. Primary antibody against IκB‐α (4814) was purchased from Cell Signalling Technology. Primary antibody against Toll‐like receptor 4 (TLR4) (AF7017) was purchased from Affinity. PolyG (P4404) and LPS (L2880) was purchased from Sigma.

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).

Western blot analyses were performed with iHEK cell samples. Approximately 10 µg of protein preparations were loaded on precast NuPAGE 4%–12% Bis‐Tris gels (Invitrogen) for SDS‐PAGE analyses. Gels were subsequently transferred onto nitrocellulose membrane and blocked overnight at 4°C with 10% nonfat dry milk. Membranes were then probed for 1 hr at room temperature with α‐MSI1 (1:1,000, ab52865 Abcam), α‐MSI2 (1:1,000, ab76148 Abcam), Pan‐Tau (Tau13—1:10,000, MMS‐520R Covance), GAPDH (1:1,000, ab9485 Abcam), LaminB1 (1:1,000, ab133741 Abcam), and Histone3 (1:1,000, ab201456 Abcam) antibodies diluted in 5% nonfat dry milk. α‐MSI1 immunoreactivity and α‐MSI2 immunoreactivity were detected with an HRP‐conjugated anti‐rabbit IgG (1:6,000, GE Healthcare). Tau13 immunoreactivity and GAPDH immunoreactivity were detected using an anti‐mouse IgG (1:6,000, GE Healthcare) diluted in 5% milk. ECL plus (GE Healthcare) was used to visualize the bands. LaminB1 and GAPDH were used to normalize and quantify the amount of nuclear and cytoplasmic proteins, respectively. The compartment extraction was conducted with Qproteome Cell Compartment Kit (Qiagen, #37502), and nuclear, cell membrane, and cytoplasmic proteins were isolated and preserved for Western analysis.

SKM-1cells were treated with VEM (20 µM), or Bortezomib (BOR, 10 nM), for 48 h. Meanwhile, VEM, at a concentration of 5 µM, or BOR, at a concentration of 4 nM, was added to 1 million MOLM-13 cells for 48 h. AML cell lines were collected and washed three times with phosphate buffer saline (PBS), then blocked in 4% paraformaldehyde, and 0.1% Triton X-100 at room temperature for 1 h. Then AML cell lines were incubated with primary antibody overnight at 4 °C. Primary antibodies were used: TRF2 (Rabbit, Abcam-ab108997), Lamin B1 (rabbit, Abcam-ab133741). AML cell lines were then washed three times in PBS and incubated with secondary antibodies for 1 h at room temperature. The following secondary antibodies were applied in our study: FITC Goat Anti-Rabbit IgG (AS011). Finally, AML cell lines were washed three times (each time for 5 min) with PBS and then incubated with CrystalMount with DAPI for 10 min. Fluorescent images were captured by using a Leica confocal microscope (Leica TCS SP8 SR).