Anti flag tag

Cells with stable overexpression or knockdown of USP39 were collected by RIPA buffer. Immunoprecipitation was conducted with anti-HA (Abcam) or anti-SRPK1 (Santa Cruz) or anti-Pan-phospho-SR (Santa Cruz). By incubation with protein A agarose (Santa Cruz), the antibodies were removed. Proteins were prepared and separated by 10% SDS-PAGE. The interaction between USP39 and SRPK1/SRSF1 was analyzed by Western blot using anti-Flag tag (Abcam) or anti-SRSF1 (Abcam).

For IHC, HCC tissue microarrays analysis (TMA) slides were purchased from Alenabio (Xi'an, China). The primary antibodies used in IHC were anti-p-p38 (Cell signaling technology (CST), Boston, MA, USA, #4511), anti-p38 (Abcam, Hongkong, China, #ab31828), anti-p-MKK3/6 (CST, #9236), anti-MKK3 (Epitomics, Burlingame, CA, USA, #1728), anti-MKK6 (Epitomics, #1640), anti-YAP (CST, #12395), anti-c-Jun (CST, #2315), anti-c-Myc (Epitomics, #1472), anti-ki67 (Abcam, #ab15580), anti-AFP (Abcam, #ab46799) and anti-SIRT1 (for mSIRT1, CST, #2028; for hSIRT1, Abcam, #ab32441). For IF, the primary antibodies used were anti-p-p38 (for mp-p38, CST, #4511; for h-p-p38, Abcam, #ab45381), anti-p38 (Abcam, #ab31828), anti-p-MKK3/6 (CST, #9236) and anti-FLAG-tag (CST, #8146). For WB, the primary antibodies used were anti-GAPDH (CST, #5174), anti-p-p38 (CST, #4511), anti-p38 (Abcam, #ab31828), anti-SIRT1 (for mSIRT1, CST, #2028; for hSIRT1, Abcam, #ab32441), anti-p-MKK3/6 (CST, #9236), anti-MKK3 (Epitomics, #1728), anti-MKK6 (Epitomics, #1640), anti-FLAG-tag (CST, #8146), anti-AFP (Abcam, #ab46799) and anti-YAP (CST, #12395). All IHC, IF and WB experiments were performed conventionally and the protocols are available elsewhere.

The normal MOVAS cells or MOVAS cells with PPARγ-knockdown were treated with Ang II+Bleo for 5 days and were lysed in NP-40 buffer with protease inhibitor cocktail and the crude lysates were cleared of insoluble debris by centrifugation at 12,000 × g. The lysates were immunoprecipitated with normal IgG or primary antibodies, including anti-FTH1 (#4393, Cell Signaling Technology), anti-HA-tag (#ab9110, Abcam) and anti-Flag-tag (#ab205606, Abcam) in a rotator (Scilogex, Rocky Hill, CT) at 4 °C overnight. The 20 μl protein A/G PLUS-Agarose beads (#sc-2003, Santa Cruz Biotechnology) were added into the 200 μl homogenates or lysates and incubated for 4 h with gentle agitation. The beads were washed 3 times with the lysis buffer and boiled with 10 μl loading buffer. The beads were removed by centrifugation (5 min at 12,000 × g). The supernatant fraction was collected and used for immunoblotting.